4. MATERIALS AND METHODS 4.1 Chemicals and reagents...
Transcript of 4. MATERIALS AND METHODS 4.1 Chemicals and reagents...
Materials and methods
42
4. MATERIALS AND METHODS
4.1 Chemicals and reagents used
Atomic absorption spectrometer (AAS) standard metal solutions of magnesium,
manganese, calcium, copper, zinc, silicon, lead, arsenic, mercury, cadmium, nickel,
chromium and aluminium were procured from Sigma Aldrich limited, Mumbai,
India. The metal solutions were diluted with deionised water to produce working
standards of 1000 ppm solutions and stored. Acrylamide, bisacrylamide, tris base,
ammonium persulphate, sodium dodecyl sulphate, mercaptoethanol, bromophenol
blue, glycine, coomassie brilliant blue G250, coomassie brilliant blue R250, bovine
serum albumin, TEMED, agarose, ethidium bromide, isoamyl alcohol, 2,4,6-
tripyridyl-s-triazine (TPTZ), dinitrophenyl hydrazine (DNPH), 2,6-dichlorophenol
indophenols were procured from Sigma Aldrich limited, Mumbai, India. Prestained
broad range SDS-PAGE standard and LA 393-5MT Dialysis membrane 70, flat
width 29.31mm, diameter 17.5mm, capacity approx. 2.41mL/cm were obtained
from Bio-Rad laboratories, Hercules, CA. Master mix, ITS1 forward primer, ITS4
reverse primer, pGEM-T easy vector, 1 kilobase pair (kb) DNA ladder were
procured from Bangalore Genei Pvt. Ltd., Bangalore, India. 3-(4, 5-dimethyl thiazol–
2–yl)–5–diphenyl tetrazolium bromide (MTT) was obtained from Acros Organics,
New Jersy, USA.
4.2 Instruments used
i. Rotary evaporator: Rota vapor R-205, Buchi Laboratory Equipments,
Switzerland.
ii. Muffle furnance: Scientec, Genuine equipment manufacturers, Coimbatore,
India
iii. Atomic absorption spectrometer: Shimadzu AA 6300 flame atomic
absorption spectrometer, Tokyo – Japan, equipped with a deuterium
background corrector and a hydride vapour generator for analysis of arsenic
Materials and methods
43
and mercury. Hollow cathode lamps of specific metals were used as a
radiation source.
iv. HPLC: Shimadzu, liquid chromatographic system equipped with LC20AT
solvent delivery system (pump), fluorescence detector and auto sampler
with 100µL loop volume was used. Class VP 6.01 data station was used for
data collection and processing.
v. Matrix Assisted Laser Desorption Ionisation: MALDI-ToF/ToF MS, Bruker
Daltonics, Ultraflex III, Germany.
vi. PCR: Eppendorf Master cycler, Germany
vii. DNA document analyser: Alpha Innotech Corporation, San Leandro,
California.
viii. Systronics pH meter
ix. Spectrophotometer: Shimadzu 160-A UV-VIS, Koyota, Japan.
x. Elisa reader : Thermo Multiskan, EX, USA.
4.3 Plant collection, authentication and extraction
The plant, Spergula arvensis Linn., was collected from in and around
Udhagamandalam in the month of August 2009 and was authenticated by
Dr.N.Selvaraj, Professor and Head, Horticulture Research Station (TNAU),
Udhagamandalam, The Nilgiris, Tamilnadu. A voucher specimen with number,
TIFAC 05, was deposited at the herbarium of JSS College of Pharmacy,
Udhagamandalam.
Processing of the sample
Approximately 5kg of the plant sample was collected and dried. Healthy plants
were removed and washed thoroughly with distilled water until no foreign
material remained. Some of the samples were dried using tray drier for 3h. The
dried samples were stored in a cupboard in capped bottles kept in dessicator and
used when required.
Materials and methods
44
Extraction
The dried plant material was powdered and successively extracted with petroleum
ether, chloroform, ethyl acetate and methanol in a Soxhlet extractor for 18–20h. The
extracts were concentrated to dryness in a rotavapor under reduced pressure and
controlled temperature (40-50°C). The nature and yields of the extracts were noted.
All the extracts were stored in a refrigerator at 4°C for further use.
4.4 Preliminary phytochemical screening of the sample
The dried plant sample obtained was subjected to preliminary phytochemical
screening. (Harborne, 1984; Kokate 2002; Raaman N., 2006).
4.5 Quantitative nutrient analysis
Proximate analysis
Proximate analysis is a technique that separates and identifies categories of
compounds in a mixture like moisture, ash content, starches, reducing sugars,
proteins, fats, esters, free acids, etc. This method of analysis helps to know about the
energy contained in the sample. The analysis was carried out as per the procedure
given by Sadasivam and Manickam, 2009.
Determination of total ash
About 3g of the powdered plant was accurately weighed in a silica crucible which
was previously ignited and weighed. The powder was spread as a fine even layer at
the bottom of the crucible. The crucible was incinerated at a temperature not
exceeding 450ºC until free from carbon. The crucible was cooled and weighed and
the procedure was repeated to get a constant weight.
Determination of moisture content
The sample material was taken in a flat-bottom dish and kept overnight in an air
oven at 100–110ºC and weighed. The loss in weight was regarded as a measure of
moisture content.
Materials and methods
45
Estimation of crude fat
The moisture free sample (2g) was extracted with petrol in a Soxhlet extractor. It
was then heated on a sand-bath for about 6h till a drop taken from the drippings
left no greasy stain on the filter paper. After boiling with petrol, the residual petrol
was filtered using Whatmann no. 40 filter paper and the filtrate was evaporated in a
pre weighed beaker. Increase in weight of the beaker gave the amount of crude fat.
Estimation of crude protein
The crude protein was determined using the micro Kjeldahl method. The oven-
dried material (2g) was taken in a Kjeldahl flask and 30mL of con H2SO4 was added
followed by 10g potassium sulphate and 1g of copper sulphate. The mixture was
then heated first gently and then strongly till the frothing ceased. When the solution
became colourless, it was heated for another hour, allowed to cool, diluted with
distilled water and transferred to a 800mL Kjeldahl flask by washing the digestion
flask. Three or four pieces of granulated zinc, and 100 mL of 40% caustic soda were
then added and the flask was connected with the splash heads of the distillation
apparatus. Dil H2SO4 (0.1N, 25mL) was taken in the receiving flask and distilled.
After two-thirds of the liquid distilled out, it was tested for the completion of the
reaction. The flask was removed and titrated against 0.1N caustic soda solution
using methyl red indicator for the determination of Kjeldahl nitrogen. The crude
protein content was calculated.
Estimation of crude fiber
The crude fiber, was determined by treating the moisture and fat-free material, first
with 1.25% dilute acid and then with 1.25% alkali, thus imitating the gastric and
intestinal action in the process of digestion. Then 2g of the moisture and fat-free
material was treated with 200mL of 1.25% H2SO4. After filtration and washing, the
residue was treated with 1.25% NaOH. It was then filtered, washed with hot water
and then 1% HNO3 and again with hot water. The residue was ignited and the ash
weighed. Loss in weight gave the weight of the crude fiber.
Materials and methods
46
Estimation of phenols
The plant sample (1g) was homogenized in 10mL of 80 per cent methanol with
pestle and mortar and agitated for 15 min at 70ºC. One mL of the methanolic extract
was added to a mixture of 5mL of distilled water and 250µL of Folin Cicalteau
reagent (1N) and the solution was kept at 25ºC. After 3 min 1mL of saturated
solution of sodium carbonate and one mL of distilled water was added and the
reaction mixture was incubated for 1h at 25ºC, when a blue colour developed. The
absorbance of the blue colour was measured using UV-Visible Spectrophotometer at
726 nm.
Estimation of carbohydrates
Carbohydrates are the important components of storage and structural materials in
plants. They exist as free sugars and polysaccharides. The basic units of
carbohydrates are the monosaccharides which cannot be split by hydrolysis into
simpler sugars. The carbohydrate content can be measured by hydrolyzing the
polysaccharidres into simple sugars by acid hydrolysis and estimating the resultant
monosaccharides. In the present study the carbohydrate was estimated by the
phenol sulphuric acid method.
Principle
In hot acidic medium, glucose gets dehydrated to hydromethyl furfural. This forms
a green coloured product with phenol and has an absorption maximum at 490nm.
Procedure
The sample (100mg) was taken in a boiling tube. It was then hydrolysed with 5mL
of 2.5N HCl, kept in a boiling water bath for 3h and cooled to room temperature.
The solution was neutralized with solid sodium carbonate until the effervescence
ceased. The volume was made up to 100mL and centrifuged. The supernatants
(0.5mL and 1mL) were collected and analysed. The glucose solutions (0.2, 0.4, 0.6,
0.8 and 1mL) were pipetted into a series of test tubes (working standards). Then the
Materials and methods
47
volume in all the tubes was made up to 1mL with distilled water. Phenol solution
(1mL) and 96% sulphuric acid (5mL) were then added to each tube. Water (1mL)
served as the blank. The contents in the tube were mixed well and after 10 min they
were placed in a water bath at 25-30ºC for 20 min and the absorbance was read at
490nm. The amount of the total carbohydrate present in the sample solution was
determined using the standard graph.
Estimation of cellulose
Principle
Cellulose undergoes acetolysis with acetic/nitric acid forming acetylated
cellodextrins which get dissolved and hydrolysed to form glucose molecules on
treatment with 67% H2SO4. This glucose molecule is dehydrated to form
hydroxymethyl furfural which forms a green coloured product with anthrone and
the colour intensity is measured at 630nm.
Procedure
Acetic acid (3mL) was added to a known amount (1g) of the sample in a test tube
and mixed in a vortex mixer. The tube was placed in a water bath at 100ºC for 30
min and cooled. The contents were then centrifuged for 15-20 min and the
supernatant was discarded. The residue was washed with distilled water and 10mL
of 67% H2SO4 was added to it and allowed to stand for 1h. The solution (1mL) was
diluted to 100mL. To 1mL of this diluted solution, 10mL of anthrone reagent was
added and mixed well. The tubes were heated in a boiling water bath for 10 min
and cooled. The colour was measured at 630nm. A blank was set with anthrone
reagent and distilled water. Cellulose (100mg) was taken in a test tube and the same
procedure was followed for the standard. A series of volumes (0.4-2mL,
corresponding to 40-200µg of cellulose) were taken and the colour developed was
read at 630nm.
Materials and methods
48
Estimation of free fatty acid
A small quantity of free fatty acid is usually present in oils along with triglycerides.
The free fatty acid content is known as acid number/acid value. It increases during
storage. The keeping quality of oil, therefore, relies upon the free fatty acid content.
Principle
The free fatty acid in oil is estimated by titrating it against KOH in the presence of
phenolphthalein indicator. The acid number is defined as the mg KOH required to
neutralize the free fatty acid present in 1g of sample. The free fatty acid content is
expressed as oleic acid equivalents.
Procedure
The oil (1g) was dissolved in 50mL of a neutral solvent in a 250mL conical flask and
a few drops of phenolphthalein was added to it. It was then titrated against 0.1N
potassium hydroxide. The contents were shaken vigorously until a pink colour
obtained persisted for fifteen seconds.
Titre value x Normality of KOH
Acid value (mg KOH/g) = X 56.1 Weight of the sample (g)
The free fatty acid was calculated as oleic acid using the equation,
1mL N/10 KOH = 0.028g oleic acid.
Note: To find out the exact strength of KOH, 0.1N oxalic acid solution (630mg in 100
mL water) was prepared and titrated against KOH with phenolphthalein as the
indicator. The strength of KOH was calculated using the formula V1N1 = V2N2.
Total free amino acid
The amino acids are colourless ionic compounds that form the basic building blocks
of proteins. Apart from being bound as proteins, amino acids also exist in free form
in many tissues and are known as free amino acids. They are mostly water soluble
Materials and methods
49
in nature. Very often in plants during disease conditions, the free amino acid
composition exhibits a change and hence, the measurement of total free amino acid
gives the physiological and health status of the plants.
Principle
Ninhydrin is a powerful oxidizing agent, decarboxylates the α-amino acids and yields an
intensely coloured bluish purple product which is colorimetrically measured at 570nm.
Procedure
Extraction of amino acids
The leaf sample (500mg) was ground with a small quantity of acid washed sand and
5mL of 80% ethanol was added to this homogenate and centrifuged. The supernatant
obtained was saved and the extraction was repeated twice and the supernatants were
pooled. The volume of the sample was reduced by evaporation and the extract was used
for the quantitative estimation of total free amino acid. To 0.1mL of the extract, 1mL of
ninhydrin solution was added and the volume was made up to 2mL with distilled
water. The test tube was heated in a boiling water bath for 20 min. To this 5mL of the
diluent mixture was added and the contents were mixed. After 15 min the intensity of
the purple colour formed was read against a reagent blank in a colorimeter at 570nm.
Standard
Leucine (50mg) was dissolved in 50mL of distilled water in a volumetric flask (stock
solution). Stock solution (10mL) was taken and diluted to 100mL in another
volumetric flask (working standard). A series of volume from 0.1-1mL of the
working standard solution gave a concentration range 10-100µg. The same
procedure as that of the sample was followed and read the colour at 570nm. The
total free amino acids in the sample was determined by a standard curve using
absorbance vs concentration expressed as percentage equivalent of leucine.
Materials and methods
50
Estimation of tannins
Tannin content in the leaf samples were estimated following the Folin-Dennis
method (Oberbacher and Vines ,1963).
Standard tannic acid solution
Tannic acid (100mg) was dissolved in 100mL of distilled water. The working
standard solution was prepared by diluting 5mL of the stock solution to 100mL
with distilled water.
Extraction of tannin
The powdered leaf material (250mg) was weighed and transferred to a 50mL
conical flask to which 10mL of water was added. The flask was heated gently and
boiled for 30 min. The flask was then cooled and the contents were centrifuged at
5000 rpm for 20 min, the supernatant was collected and the volume was made up to
10mL. The sample extract (0.5mL) was transferred into test tube and the volume
was made up to 7.5mL with water. To this, 0.5mL of Folin-Denis reagent and 1.0mL
of sodium carbonate solution were added and shaken well. The absorbance was
read at 700 nm after 30 min. A blank was run with water instead of the sample. The
tannin content was calculated as tannic acid equivalents from the standard graph.
Estimation of lycopene
Lycopene is responsible for the red colour of tomato and the fleshy part of water
melon. It is a carotene having the formula C40H56. Though, it has no nutritional
value, its contribution to the colour of tomato has a great role in consumer
acceptability.
Principle
The carotenoids in the sample are extracted in acetone and then taken in petroleum
ether. Lycopene has absorption maxima at 473nm and 503nm. One mole of
lycopene when dissolved in one litre light petroleum (40 - 60ºC) and measured in a
Materials and methods
51
spectrophotometer at 503nm in 1cm light path gives an absorbance of 17.2 x 104. A
concentration of 3.1206µg lycopene/mL, therefore, gives unit absorbance.
Procedure
The plant sample (2g) was taken and grinded well to a smooth consistency in a
blender. About 1g of the ground sampled was washed accurately. The sample was
then repeatedly extracted with acetone using pestle and mortar until the residue
became colourless. The acetone extracts were pooled and transferred to a separating
funnel containing about 20mL petroleum ether and mixed gently. About 20mL of
5% sodium sulphate solution was then added. The separating funnel was gently
shaked. The volume of petroleum ether reduced during this process because of
evaporation. More of petroleum ether (20mL) was then added to the separating
funnel for a clear separation of two layers. Most of the colour was noticed in the
upper petroleum ether layer. The two phases were separated (the lower aqueous
phase was colourless). The petroleum ether extracts were pooled and washed once
with a little distilled water. The washed petroleum ether extract containing
carotenoids was poured into a brown bottle containing anhydrous sodium sulphate
(10g) and kept aside for 30 min. It was then poured into a 100mL volumetric flask
through a funnel containing cotton wool. The sodium sulphate slurry was washed
with petroleum ether until it was colourless and the washings transferred into the
volumetric flask. The volume was made up and the absorbance was measured in a
spectrophotometer at 503nm using petroleum ether as blank.
Calculation
Absorbance (1 unit) = 3.1206 µg lycopene/ mL
31.206 x absorbance Lycopene (mg) in 100g sample = Weight of sample (g)
Materials and methods
52
Estimation of starch
Starch is an important polysaccharide. It is the storage form of carbohydrate in
plants and abundantly found in roots, tubers, stems, fruits and cereals. Starch,
which is composed of several glucose molecules, is a mixture of two types of
components, namely amylase and amylopectin. Starch is hydrolysed into simple
sugars by dilute acids and the quantity of simple sugars is measured
colorimetrically.
Principle
The sample is treated with 80% alcohol to remove sugars. Starch is then extracted
with perchloric acid. In hot acidic medium, starch is hydrolysed to glucose and
dehydrated to hydroxymethyl furfural. This compound forms a green coloured
product with anthrone.
Procedure
The sample (0.1g) was homogenised in hot 80% ethanol to remove sugars,
centrifuged and the residue was retained. The residue was washed repeatedly with
hot 80% ethanol till the washings did not give colour with anthrone reagent. The
residue was dried well over a water bath. To the residue, 5mL of water and 6.5mL
of 52% perchloric acid were added and extracted at 0ºC for 20 min, centrifuged and
the supernatant was saved. The extraction was repeated using fresh perchloric acid
and centrifuged, again the supernatants were pooled. The supernatant (0.1mL) was
pipetted out and made up the volume to 1mL with distilled water. Standards were
prepared by taking 0.2, 0.4, 0.6, 0.8 and 1mL of the working standard and the
volume made up to 1mL in each tube with water. Anthrone reagent (4mL) was then
added to each tube and heated for 8min in a boiling water bath. The solution was
rapidly cooled and the intensity of the green to dark green colour formed was read
at 630nm.
Materials and methods
53
Calculation
Glucose content in the sample was obtained using the standard graph and
multiplying the value by a factor 0.9 to arrive at the starch content.
Estimation of total antioxidants
Total antioxidant activity is measured by ferric reducing antioxidant power (FRAP)
assay (Benzie and Strain, 1999). FRAP assay uses antioxidants as reductants in a
redox linked colorimetric method.
Principle
At low pH, reduction of ferric tripyridyl triazine (Fe 3+ TPTZ) complex to ferrous
form (which has an intense blue colour) can be monitored by measuring the change
in absorption at 593nm. The reaction is non-specific, in that any half reaction that
has a lower redox potential, under reaction conditions, than that of ferric-ferrous
half reaction, will drive the ferric to ferrous (Fe 3+ to Fe 2+) ion formation. The
change in absorbance is therefore, directly related to the combined or total reducing
power of the electron donating antioxidants present in the reaction mixture.
Reagent preparation
Acetate buffer 300mM (pH 3.6)
i. Sodium acetate trihydrate (3.1g) was weighed and 16mL of glacial acetic acid
was added and made upto 1L with distilled water.
ii. 10mM TPTZ (2,4,6-tripyridyl-s-triazine) was dissolved in 40mM HCl
iii. 20mM FeCl3.6H2O were taken
The working FRAP reagent was prepared by mixing solution (i), (ii) and (iii) in the
ratio 10:1:1 at the time of use.
Standard : 1000µM Ascorbic acid
Materials and methods
54
Procedure
The sample (100µL) was mixed with 3mL of working FRAP reagent and the
absorbance was read at 593nm at 0 min after vortexing. The samples were placed at
37ºC in water bath and the absorption was again measured after 4 min. Ascorbic
acid standards (100µM-1000µM) were processed in the same way.
Calculation
Change in absorbance of sample from 0-4 min FRAP value of = X FRAP value of the Sample (µM) Change in absorbance of standard from 0-4 min std (1000µM)
Estimation of vitamin C
Ascorbic acid, otherwise known as vitamin C, is an antiscorbutic. It is present in
gooseberry, bittergourd, etc., in high amounts. Generally it is present in all fresh
vegetables and fruits. Vitamin C is a water soluble and heat liable vitamin. Ascorbic
acid was determined colorimetrically. Dehydroascorbic acid alone reacts
quantitatively and not the other reducing substances present in the sample. This
method thus gives an accurate analysis of ascorbic acid content than the dye (2,6-
dichlorophenol indophenol) method.
Principle
Ascorbic acid is first dehydrogenated by bromination. The dehydroascorbic acid is
then reacted with 2,4 dinitrophenyl hydrazine to form osazone and dissolved in
sulphuric acid to give an orange-red colour solution which is measured at 540nm.
Extraction
The sample (1g) was grinded in a mortar and pestle in 25-50mL of 4% oxalic acid
solution. This solution was filtered and the filtrate was collected. The collected
filtrate (10mL) was transferred to a conical flask and bromine water was added
dropwise with constant stirring. The enolic hydrogen atoms in ascorbic acid were
Materials and methods
55
removed by bromine. The extract turned orange yellow due to excess bromine. The
bromine was expelled by blowing in air. This was made upto 25mL with 4% oxalic
acid solution. Similarly the ascorbic acid stock solution was converted into dehydro
form by bromination.
Procedure
The standard dehydroascorbic solution (10-100µg) was pipetted out into a series of
test tubes. The brominated sample (0.1-2mL) was also pipetted out. The volume
was made upto 3mL in each tube by adding distilled water. Dinitrophenyl
hydrazine (DNPH) reagent (1mL) was added to which 2 drops of thiourea was also
added. Distilled water (3mL), 1mL DNPH reagent and 2 drops of thiourea were
taken as blank. The contents in the tubes were mixed thoroughly and incubated at
37ºC for 3h. The orange red osazone crystal formed was dissolved by adding 7mL
of 80% sulphuric acid. The absorbance was read at 540nm. For calculating the
ascorbic acid content in the sample, a graph was plotted with ascorbic acid
concentration against absorbance.
Estimation of vitamin A
Vitamin A (retinol) is a fat-soluble vitamin. It is important for proper vision.
Vitamin A is supplied to the body in the form of its precursor, β-carotene, which is
present in fruits, vegetables, greens, etc. Vitamin A is not stored in the body when
consumed abundantly.
Principle
Vitamin A and its palmitate forms blue colour with trichloroacetic acid (TCA)
which is proportional to its concentration measured at 620nm.
Extraction
The sample (1g) was grinded to a fine paste and 1mL of saponification mixture (2N
KOH in 90% alcohol) was added to it. The tube was refluxed gently for 20 min at
60ºC. The tube was cooled at room temperature and 20mL water was added and
Materials and methods
56
mixed well. Vitamin A was extracted with petroleum ether in a separating funnel,
twice. The organic layers were pooled and anhydrous sodium sulphate was added
to remove the moisture for 30-60 min. The ether extract (5mL) was evaporated to
dryness at 60ºC. The dried residue was dissolved in 1mL of chloroform.
Procedure
Aliquots of the standard vitamin A concentrations ranging from 1.5-7.5µg, acetate
were pipetted out into a series of test tubes. The volumes were made upto 10mL
with chloroform. Trichloroacetic (2mL) solution was added by rapidly mixing the
contents of the tube. The absorbance was read immediately at 620nm with a
spectrophotometer. The absorbance of the sample was also determined in the same
manner. The amount of vitamin A was calculated using a standard graph plotting
absorbance against vitamin A concentration.
Estimation of β-carotenes
Carotenoids, the tetraterpenoid (C40) compounds, are ubiquitous in plants. These
terpenoids existing as hydrocarbons (carotenes) or oxygenated derivatives, are
accessory pigments in photosynthetic systems and give characteristic colour to
plant parts, particularly flowers and fruits. Carotenes occurring in different
chemical forms have characteristics features and functions. Their levels are altered
during physiological and pathological conditions.
Principle
The total carotenoids are extracted and partitioned in organic solvents on the basis
of their solubility.
Procedure
The plant was finely cut with scissors. The weighed sample (2g) was placed in a
high speed blender. Acetone (40mL), hexane (60mL) and MgCO3 (0.1g) were added
and blended for 5 min. The residue was allowed to settle and decanted into a
separator. The residue was washed twice with 25mL portions acetone then with
Materials and methods
57
25mL hexane and the extracts were combined. The acetone, present in the extracts,
was removed by washing the extract with five 100mL portions water. The upper
layer was transferred to a 100mL volumetric flask containing 9mL acetone and the
volume diluted with hexane. In a chromatographic tube a pack of magnesia-
diatomaceous earth (1:1) was activated. The column was prepared by placing a
small glass plug inside the tube and the adsorbent was added to 15cm depth, the
tube was attached to the suction flask and vacuum was applied. Sodium sulphate
(anhydrous) (1cm layer) was placed. Vacuum was continuously applied to the flask
and the extract was poured. Acetone-hexane mixture (1:9) (50mL) was used and the
chromatogram was developed. Visible carotenes were washed through the
adsorbent. The column at the top was covered with the solvent during the process.
The entire eluate was collected and transferred to a 100mL volumetric flask to
which acetone-hexane mixture was added for dilution. The instrument was
calibrated with different concentrations of high purity β-carotene. The absorbance
of the sample solution was determined with a spectrophotometer. Carotene content
in the sample was calculated using the calibration curve.
Elemental analysis by Atomic Absorption Spectrophotometric (AAS) method
(atomic adsorption cook book).
All the reagents used for the study were of analytical grade. Deionised water was
used for all the dilutions. Nitric acid, perchloric acid and hydrogen peroxide were
of supra pure quality. All the plastic materials and glasswares were cleaned by
soaking in dilute nitric acid for 24h and rinsed with distilled water, followed by
deionised water prior to use.
The calibration curves for the analyte ions were drawn after setting the various
parameters of flame atomic absorption spectrometer including the wavelength, slit
width, lamp current, flame type, fuel gas flow rate, support gas flow rate and the
burner height at optimum levels. These instrumental parameters were tabulated.
Materials and methods
58
Linearity
The standard solutions of the analytes, namely sodium, potassium, magnesium,
manganese, calcium, copper, zinc, silicon, lead, arsenic, mercury, cadmium, nickel,
chromium, iron, palladium and aluminium. The solutions were diluted to 1000 ppm
solutions and stored in light resistant containers. The standard solutions for
calibration curve were prepared by diluting the stock solution with deionised
water.
Limit of detection and Limit of quantification
The limit of detection and the limit of quantitation of the developed method were
determined by using the formula LOD = 3Sbl/slope and LOQ = 10Sbl/slope where
Sbl is the standard deviation of blank measurements and slope of the calibration
curve.
Accuracy
The accuracy of the method was determined by recovery experiments. The recovery
of the optimised extraction procedures for the estimation of major trace minerals
and heavy metals in the plant sample were determined at single level by adding a
known quantity of the standard mineral and metal to the plant sample of pre
analysed sample and the mixtures were analysed.
Preparation of samples
Dry ashing technique was used for the digestion of the plant materials in order to
remove the organic matters (Barbara, 2003). The experiment was performed in
triplicate.
The shade dried plant material (5g) was ground into coarse powder. The ground
powder (1g) was accurately weighed and placed in clean and dry quartz beaker.
The quartz beaker was placed in a muffle furnace and the temperature was raised at
a rate of 50ºC/h until it reached 500ºC. The samples were heated at 500ºC for 24h to
conduct ashing. After the ashing was complete the samples were cooled to room
Materials and methods
59
temperature in a dessicator. After cooling 2 drops of con HNO3 were added to the
ashes for dissolving. The samples were then diluted with deionised water, filtered
using Whatmann filter paper No.42 impregnated with HNO3 and the volume of the
clear solution was made to 100mL with deionised water. The blanks were also
prepared. The above solutions were appropriately diluted prior to analysis for the
estimation of minerals and heavy metals. The recovery of the minerals and heavy
metals were carried out, percentage recovery was established and tabulated.
4.6 Amino acid analysis
Sample preparation
The dried plant sample was analyzed in duplicate for amino acids. The sample
(5mg) was weighed and placed in a 2mL ampoule, to which 0.45mL of 6N HCl were
added. The ampoules were evacuated, sealed and kept for 24h digestion.
Preparation of reagents
Mobile Phase A [pH : 3.2 (The pH was adjusted with perchloric acid)]
Sodium Citrate : 19.6g (the crystals were dissolved completely in
500mL water)
Ethanol : 70mL
Perchloric acid : 16.6mL
The total volume was made up to 1L
Mobile Phase B [pH : 10 (The pH was adjusted with 4M Sodium hydroxide)]
Sodium Citrate : 39.2g (dissolved completely)
Boric acid : 8.26g (dissolved completely)
4N Sodium hydroxide :20mL
The total volume was made upto to 666mL.
Materials and methods
60
Mobile Phase C
Sodium hydroxide : 1.3g
The total volume was made upto 166mL.
The prepared solution A, B and C were filtered through 0.45µm membrane filter, to
avoid contamination with ammonia.
Preparation of Reaction Reagent
Carbonic Acid Buffer pH: 10
Sodium carbonate : 40.7g
Boric Acid : 13.56g
Potassium Sulphate : 18.18g
The volume was made upto 1L with water
Reagent A: O-Phthaladehyde (OPA)
O-Phthaladehyde : 160mg dissolved in 2.8mL of ethanol.
50mL of carbonic acid buffer was added
Mercaptoethanol : 0.4mL
Brij : 0.8mL (5g of Brij was dissolved in 25mL of water
and the bottle immersed in warm water. After cooling, the volume was made
up to 50mL)
Finally the volume was made up to 200mL with carbonic acid buffer
Reagent B: Sodium Hypochlorite
80µL of sodium hypochlorite in 200mL of carbonic acid buffer.
Materials and methods
61
Sample Diluent pH 2.2 (The pH was adjusted with perchloric acid)
Trisodium citrate : 17.4g
Caprylic acid : 0.1mL
Perchloric acid : 11mL
The volume was made up to 1L
Procedure
The standard and sample solutions were analyzed by reverse phase high pressure
liquid chromatography (RP-HPLC) method using fluorescence detector for the
quantification of amino acids. A shimadzu LC20AT system equipped with a
quarternary pump and a fluorescence detector was used. A gradient elution for 70
min was performed using a stationary phase of Hibar C18 column (250 x 4.6mm i.d.,
5µ) and a mobile phase A consisting of sodium citrate buffer (pH adjusted with
perchloric acid) and ethanol (30:70) : mobile phase B consisting of sodium citrate
buffer (pH 10 adjusted with 4M sodium hydroxide : mobile phase C consisting of
2M sodium hydroxide). The mobile phase was pumped at a flow rate of
0.5mL/min.
Precolumn derivatisation of the sample and standard solutions were performed
using o-phthalaldehyde reagent. The retention times of the various amino acids
were determined using an amino acid calibration mixture. Quantitation of
individual amino acids was achieved by monitoring the absorption of the column
eluate at 348 and 450nm and comparing the areas under the individual peaks with
those of the corresponding amino acid standards. The essential acids were then
calculated using the formula,
Essential amino acid score= g of essential amino acid in 100g of test protein/g of
essential amino acid in 100g of FAO/WHO (1991) reference pattern x 100 (Vadivel
and Janarthanan, 2001).
Materials and methods
62
4.7 Molecular authentication
Genomic DNA extraction
Principle
In prokaryotes, the DNA is double stranded and circular and is found throughout
the cytoplasm. The cell membranes must be disrupted in order to release the DNA
in the extraction buffer. Nucleic acids are generally precipitated using ethanol,
isopropanol or PEG. Ethanol precipitation is simple, rapid and quantitative. It
precipitates even nanogram quantities of DNA and RNA.
Cetyltrimethylammonium bromide (CTAB) is a cationic detergent. The positively
charged detergent has a high affinity for the negatively charged phosphate
backbone on nucleic acid molecules and thus binds to DNA strongly and forms an
insoluble complex. CTAB solubilizes membranes, lysis the cells and precipitates cell
wall polysaccharides in presence of high salt concentration and at high
temperature. It precipitates DNA at low salt concentration and at low temperature.
Sodium dodecyl sulphate (SDS) is used to disrupt the cell membrane. DNA can be
protected from endogenous nucleases by chelating with Mg2++ ions using EDTA.
Mg2++ ions are considered as a necessary cofactor for most nucleases. Proteinase
enzyme is used to degrade the proteins in the disrupted cell soup. Phenol–
chloroform is used to denature and separate the protein from the DNA. Chloroform
is also a protein denaturant, which stabilizes the rather unstable boundary between
an aqueous phase and a pure phenol layer at the interface between the aqueous and
organic phases which are removed by centrifugation. DNA released from the
disrupted cells is precipitated by cold absolute ethanol or isopropanol.
Materials and methods
63
Preparation of the reagent
Cetyltrimethylammonium bromide (CTAB) solution
CTAB - 2g
0.1M Tris HCl (pH 8.0) – 10mL
1.4M NaCl – 8.18g
0.5M EDTA (pH 8.0) – 4mL
PVP – 1g
Mercaptoethanol – 1mL
Sodium sulphite – 1g
Procedure
The plant was ground into a fine powder using liquid nitrogen and incubated in
2mL of CTAB extraction buffer (0.1M Tris–HCl, pH 7.5, 0.7M NaCl, 10mM EDTA,
1% CTAB) at 65°C for 10 min. The mixture was treated with 750µL of chloroform :
isoamyl alcohol (24:1) and the solution was gently rotated for 10 min at room
temperature and centrifuged at 10,000 rpm for 10 min. The upper aqueous layer
(300µL) was mixed with 0.5 volume of 5M sodium chloride and 2 volumes of ice
cold ethanol, incubated at -20°C for 1h and then centrifuged at 13,000 rpm at 4°C for
10 min. The precipitated DNA was then washed with 70% ethanol and resuspended
in 300µL of TE buffer (10mM Tris HCl, pH 8.0 and 1mM EDTA). RNA was digested
with RNase (1mg/mL) at 37°C for 1h. The chloroform extraction was repeated one
more time. The DNA was precipitated by adding 100µL of 5M sodium acetate (pH
5.2) and 725µL of ice cold 100% ethanol. The pellet was washed with 500µL of ice
cold 70% ethanol, air dried and dissolved in 50µL of Tris- EDTA (TE) buffer
depending on the pellet size. The DNA concentration was determined by 1%
agarose gel.
Materials and methods
64
Polymerase Chain Reaction (PCR)
Polymerase chain reaction is a process where the two synthetic oligonucleotide
primers, which are complementary to two regions of the target DNA, are added in
the presence of excess deoxynucleotides and Taq polymerase.
Principle
The polymerase chain reaction (PCR) is an in vitro DNA amplification of target
DNA with a pair of primers and a DNA polymerase, resulting in several million
fold amplification of the target sequence within a few hours. In this way, the sample
(target DNA) is allowed to react with a pair of primers (specific for each microbe),
deoxynucleotide triphosphates, buffer and Taq DNA polymerase (heat stable DNA
polymerase). Of the primers that are complementary to the target DNA, a new
DNA strand is synthesized. During each cycle, the DNA strand is doubled. Each
cycle consists of three segments, namely denaturation step (during which the two
strands of target DNA is separated), annealing step (during which the primers
attaches to the complementary target sequences) and synthesis or extension step
(during which a new strand is synthesized with a help of dNTPs and enzyme).
Procedure
Amplification of DNA was performed in a total volume of 20µL. The reaction
mixture contained 10µL of 10X reaction buffer [500mM KCl, 15mM MgCl2, 100mM
tris HCl (pH 8.3), 0.1% w/v gelatin] Template DNA–DNA sample was added at a
concentration of 50ng/µL. Each of the forward (ITS1 5'-
TCCGTAGGTGAACCTGCGGAAGGATCATTG-3') and reverse primer (ITS4 5'-
TCCTCCGCTTATTGATATGC-3') (20pmol) was then added. dNTP mix (3µL of
5mM) was then added. The four deoxyribonucleotide triphoshate (dATP, dTTP,
dGTP, dCTP) were used at a concentration of 100mM each. 1µL of Taq polymerase
(0.5U/µL) was added to each sample. The remaining volume was made up by
sterile water. Amplification was carried out in an Eppendorf Master Cycler. Initial
denaturation was carried out at 94ºC for 4 min, followed by 35 cycles, each cycle
Materials and methods
65
consisting of denaturation of DNA for 1 min at 94ºC, annealing of the primers for 1
min at 52ºC, elongation at 72ºC for 2 min and final elongation for 10 min. The PCR
products were analysed by gel electrophoresis in a 1.2% agarose in Tris – acetate
buffer (pH 8.3) – EDTA (TAE) buffer (0.04M Tris – acetate, 0.001M EDTA, pH 8.0).
A 1000 base pair (1Kbp) ladder was used as a size standard. To visualize DNA, gels
were stained with ethidium bromide (0.1mg/L) and then photographed under
transmitted ultraviolet light using an Alpha Imager 2000 (Alpha Innotech, San
Leandro, CA, USA). For primer and plant isolate combination, the amplification
reactions were performed three times to determine the reliability and
reproducibility of the method.
DNA cloning
Competent cell preparation
Principle
E.coli cells are more likely to incorporate foreign DNA if their cell walls are altered
so that DNA can pass through more easily. Such cells are said to be competent.
Cells that are undergoing very rapid growth are made competent more easily than
cells in other stages of growth. The cells can be made competent artificially by
treating the cells with calcium chloride prior to adding DNA. The calcium
destabilizes the cell membrane and adheres to the cell surface favouring the
formation of the pores for the entry of DNA. The DNA is taken during the heat
shock step when the cells are exposed briefly at the temperature of 42ºC. Immediate
chilling on ice ensures closure of pores.
Selection of cells containing transformed DNA is enhanced by selection markers
carried by DNA. PUC series and PBR322 plasmids have ampicillin resistance factor
which enables only the transformed cells to grow on Luria Bertani – ampicillin
plate.
Materials and methods
66
The PUC plasmid also has the genes for β – galactosidase enzyme. The lacZ is a
gene that has a series of unique restrictions site that has a series of unique
restrictions site engineered into it such that the plasmid be cut within lacZ gene. If
the plasmids are the host or the plasmid encoded fragments are themselves active,
they can associate to form an enzymatically active protein. This type of
complementation is known as α – complementation.
LacZ positive bacteria, result from α–complementation can produce active β–
galactosidase enzyme which hydrolyse X–gal (5 – bromo, 4–chloro, 3–indolyl β–D–
galactoside) into a blue coloured compound and thereby appears as a blue coloured
colony in presence of X–gal and IPTG. Any plasmid which has been inserted with
DNA fragment in LacZ gene will not have a functional LacZ gene and thus will
produce white colonies which are unable to cleave X–gal.
Reagent Preparation
i. Calcium chloride (100mM)
Calcium chloride (1.47g) was dissolved in 100mL of distilled water, sterilized
and stored at 4˚C.
ii. Isopropyl thiogalactoside (IPTG) stock
IPTG (20mg) was dissolved in 1mL of distilled water.
iii. X–gal stock
X–gal (20mg) was dissolved in 1mL dimethyl formamide and stored at –
20˚C.
iv. IPTG / X – gal
IPTG stock (800µL) was added to 3.8mL of sterile water and swirled well to
mix. X–gal (400µL) stock was added and mixed till the solution was
colourless. 250µL per plate was used and the solution was stored at – 20˚C.
Materials and methods
67
Procedure
Competent preparation
The isolated colonies of E.coli was plated and inoculated into the LB broth and
incubated for overnight at 37ºC. From the overnight culture, a fresh culture was
prepared. The broth (2mL) was taken in the eppendorf tube and centrifuged at 6000
rpm for 10 min at 4ºC. The supernatant was discarded and the pellet was
suspended in 1mL of 0.1M calcium chloride. The tubes were kept in ice for 20 min.
It was then centrifuged at 6000 rpm for 10 min at 4ºC. The supernatant was again
discarded and the pellet was resuspended gently in 100µL of 0.1M calcium chloride
and 16µL of 40% glycerol. The competent cell prepared was stored at 4ºC.
Transformation
The competent cell (100µL) was taken and 3µL of plasmid pGEM-T easy vector was
added and mixed gently. The tubes were kept in ice for 30 min. It was then
subjected to heat shock at 42ºC for 1 min by keeping in the water bath. This was
kept in ice for 5 min and 600µL of LB broth was added and incubated at 37ºC for 1h.
LB ampicillin plate along with IPTG and X – gal was prepared with the antibiotic
concentration of 100µg/ml and IPTG X–gal concentration of 200µg/mL. The
inoculums was plated and incubated at 37ºC for overnight and the results were
observed.
Isolation of Plasmid DNA
Plasmids are extrachromosomal, self replicating double stranded, circular DNA
molecule present in most prokaryotes. Plasmids are responsible for antibiotic
resistant gene transfer between individual cells. The plasmids can be transferred
from one cell to another and therefore function as vectors or carriers in genetic
engineering techniques.
Materials and methods
68
Principle
Glucose provides an iso osmotic condition to prevent physical shock. The
resuspended solution‟s pH is raised to basic level with Tris to help denature DNA.
EDTA is added to protect DNA from endogenous nucleases. It is a chelating agent
that binds Mg2++ ions, which is considered a necessary cofactor for most nucleases.
The sodium dodecyl sulfate (SDS) is an ionic detergent, which dissolves the
phospholipids and protein components of cell membrane. Sodium hydroxide in the
solution denatures the plasmid and the genomic DNA into single strands.
Potassium acetate forms an insoluble precipitate of SDS / lipid / protein complex.
At this pH, the circular DNA renatures. The chromosomal DNA is trapped in the
SDS / lipid / protein precipitate. The plasmid DNA renatures into its double
stranded form and escape being trapped in the precipitate and remains in the
supernatant. Phenol chloroform is used to denature and separate the proteins from
DNA. Phenol efficiently denatures proteins and probably dissolves the denaturized
protein. Chloroform, also a protein denaturant, stabilizes the rather unstable
boundary between an aqueous phase and a pure phenol layer. The use of this
mixture also reduces the amount of aqueous solution remained in the organic phase
in order to maximize the yield. The enzyme RNAse will completely degrade the
contaminating RNA available in the DNA solution.
Preparation of reagents and medium
i. Luria Bertani broth
Tryptone - 10mg /L
Yeast extract - 5g /L
NaCl - 10g /L
Ampicillin - 10µg /mL
(pH adjusted to 7.5 adding NaOH dropwise)
Materials and methods
69
ii. Solution I
50mM glucose
25mM Tris HCl (pH 8.0)
10mM EDTA (pH 8.0)
iii. Solution II
0.2N NaOH (freshly diluted from a 10N stock)
1% SDS
iv. Solution III
5M potassium acetate - 60mL
Glacial acetic acid - 11.5mL
H2O - 28.5mL
(The resulting solution is 3M with respect to potassium and 5M with
respect to acetate)
v. Phenol chloroform mixture
Equal volumes of phenol and chloroform were mixed. The mixture
was kept on ice and added 20mL of TE buffer. The mixture was mixed
for 15 min to remove the dust on the surface layer using a pipette.
This was repeated 5 times. TE buffer (30-40mL) was added and it was
stored on ice.
vi. 95% ethanol
vii. 70% ethanol
viii. TE buffer containing DNase free RNase
10mM Tris HCl (pH 8.0)
1mM EDTA (pH 8.0)
RNase
Steps
Growing the bacteria and amplifying the plasmid – preparation of
preculture
Materials and methods
70
Harvesting and lysing the bacteria
Purifying the plasmid away from the bacterial host
Procedure
The bacterial culture was streaked on LB agar containing ampicillin (100µg/mL)
and incubated at 37ºC overnight. A single colony was picked using autoclaved
tooth pick and the inoculums were transferred into 5mL of LB broth containing
100µg/mL ampicillin and incubated at 37˚C with shaking for 8–12h (150 rpm). The
overnight culture (1mL) was transferred into 1.5mL eppendorf tube and centrifuged
for 5 min at 10000 rpm. The supernatant was discarded. Solution I (100µL) was
added to the cell pellet and vortexed in a vortex mixture so that the cells dissolved
and dispersed uniformly. It was incubated at room temperature for 5 min. Solution
II (200µL) was added and gently mixed by inverting the tube 5 times and incubated
at ice for 10 min. Ice cold solution III (150µL) was then added and gently mixed by
inverting the tube 5 times and incubated at ice for 10 min.
The tube containing the lysate was centrifuged at 10000 rpm for 10 min at 4ºC. The
supernatant was transferred into a fresh tube without disturbing the pellets. Phenol
chloroform mixture (400µL) was added and gently mixed by inverting the tube 5
times and incubated at room temperature for 5 min. The tube was centrifuged at
10000 rpm for 10 min. The supernatant was transferred into a fresh tube without
disturbing the precipitate. Absolute ethanol (1mL) was added and mixed by
inverting the tube 5 times and incubated at 20ºC for 1h for DNA to precipitate. It
was centrifuged again at 10000 rpm for 5 min and the supernatant were discarded.
The precipitate obtained was washed with 1mL of 70% ethanol. The alcohol was
completely drained off. When the pellet turned transparent, 20µL of 1X TE buffer
containing RNase enzyme was added and incubated for 1h. The presence of
plasmid DNA was checked on agarose gel electrophoresis.
Materials and methods
71
Agarose Gel Electrophoresis
Agarose gel electrophoresis is employed to quickly determine the yield and purity
of DNA isolated or check the product of PCR reaction, check progression of a
restriction enzyme digestion and to size fractionate DNA molecules which then
could be eluted from the gel. Agarose is a natural product purified from red
seaweed (Rhodophta). It is a polysaccharide of alternate 1,4 linked α-D-
galactopyranose and 1,4 linked 3,6-anhydro-α-L-galactopyranose residue and
arranged into double helix. It dissolves in water on boiling and forms a gel when
cooled to about 40ºC.
Agarose gel is the first choice for nucleic acid analysis. The gel is easily prepared,
nontoxic, optically clear (preferred for densitometric scanning and photography),
chemically inert having pores for a wide range of molecules to pass through,
available with least or minimum electroendosmosis (EEO) possessing good gel
strength.
Agarose gels have large pore size and can be used to separate macromolecules like
nucleic acids. Agarose gels are hydrocolloids, held together by hydrogen and
hydrophobic bonds. They are somewhat brittle and they break when bent. Hence,
agarose gels should be handled carefully with some form of support for the entire
gel, such as gel tray or wide spatula. The pore size and sieving characteristics of
agarose gel to a certain extent is determined by its concentration. The higher the
concentration the smaller is the pore size. Agarose gel is generally used in the
concentration range of 0.4- 4% (w/v).
Principle
The separation of DNA on the gel is carried out under an electric field applied to
the gel matrix. DNA molecules migrate towards the anode due to the negatively
charged phosphates along the backbone of the DNA. Fragments of linear DNA
migrate through agarose gels with the mobility that is inversely proportional to the
Materials and methods
72
log10 of their molecular weight. Thus the larger molecules travel at a slower speed
than the smaller ones.
Circular forms of DNA migrate in agarose distinctly from linear DNAs of the same
mass. Typically uncut plasmids will appear to migrate more rapidly than the same
plasmid when linearised. Additionally, most preparations of uncut plasmid contain
at least two topologically different forms of DNA, corresponding to supercoiled
forms and nicked circles. Several parameters like agarose concentration, voltage
applied and molecular size of DNA affect the migration of DNA.
Reagent preparation
i) Tris acetate (TAE) buffer (50X) was stocked.
Tris base (1.6M) – 242g
Glacial acetic acid – 57.1mL
0.5M EDTA (pH 8.0) – 100mL
ii) 1X TAE buffer
1mL of stock TAE buffer was diluted to 50mL with autoclaved distilled
water.
iii) Gel loading dye (1X)
Bromophenol blue – 0.25g
Xylene cyanol – 0.25g
Sucrose – 40g
The above were dissolved in 100mL of distilled water and stored at -20˚C
iv) Ethidium bromide stock
10mg of ethidium bromide was dissolved in 1mL of distilled water.
Materials and methods
73
Procedure
Agarose gel electrophoresis was performed based on the method described by
Sambrook et al. (1989) to check the quality of DNA and also to separate the products
amplified through polymerase chain reaction. 1X TAE tank buffer in 500mL
quantity was prepared to fill the electrophoresis tank and for gel preparation. In a
separate conical flask, agarose (0.8 per cent for genomic DNA and 1.5% for PCR
product) was added to 1X TAE buffer, boiled till the agarose dissolved completely
and cooled to lukewarm temperature. Ethidium bromide was added at the rate of
5L/100mL to agarose solution and was allowed to mix completely. It was then
poured into the gel mould, the comb was placed properly and allowed to solidify
for half an hour at room temperature.
After solidification, the comb was removed carefully. The casted gel was placed in
the electrophoresis tank containing 1X TAE buffer with the well near the cathode
and submerged to a depth of 1cm. Fifteen microlitre of the PCR product was mixed with
3L of 6X tracking dye and mixed well by pipetting in and out 3 times. The mixture was
loaded into the wells with the help of the micropipette. Two microlitre of 1 kbp DNA
ladder was loaded in one of the wells as a standard marker. The cathode and anode
were connected to power pack using power cord and the gel was run at a constant
voltage of 60 volts. The negatively charged DNA molecules moved towards the
anode and got separated according to their molecular weight. The power was
turned off when the marker reached the anode end and the gel was viewed in an
UV transilluminator and the banding pattern was analyzed. The sizes of the PCR
products were determined by comparison with standard 1 kbp molecular marker
(Genei, Pvt. Ltd., Bangalore, India).
Gel documentation
After the separation of the PCR products with 1.0% agarose gel, it was viewed and
photographed using Alpha imager TM1200 documentation and analysis system. The
PCR products were resolved on 2% agarose at 50V stained with ethidium bromide
Materials and methods
74
(0.5µg/mL), photographed and analyzed using gel documentation system (Alpha
Innotech Corporation, San Leandro, California).
Sequencing of ITS region
ITS region was amplified with respective ITS primers. Amplified 16S rDNA was
purified from each reaction mixture by agarose (1.2%, w/v) gel electrophoresis in
TAE buffer containing 0.5µg of ethidium bromide per mL. Colony PCR was
performed for the cloned DNA. The plasmid was isolated by conventional method.
The isolated plasmid was then sequenced.
ITS sequence analysis
To obtain the genomic sequence of the plant, vector sequence was removed using
the software Vecscreen. Vecscreen is a system for quickly identifying segments of a
nucleic sequence that may be of vector origin. This software helps researchers to
identify and remove segments of vector origin before sequence analysis or
submission. This helps us to deduce the vector contamination from the cloning
history of the sequenced DNA.
Sequence submission
The rDNA homology searches were performed using the BLAST program (Altschul et
al., 1990) through the internet server at the National Center for Biotechnology
Information (National Institutes of Health, Bethesda, USA). Sequences were compared
with the GenBank database. Sequin software was used to prepare the sequence format
for submission. The newly obtained sequences were submitted to GenBank database,
New York, USA.
4.8 Protein Extraction
The plant was homogenized with a mortar and a pestle in liquid nitrogen. The
frozen plant was placed in 500mL Scott Duran bottle and about (1:4 ratio) 0.1M
potassium phosphate buffer (pH 7.0) was added and vortexed. The homogenates
were mixed thoroughly and incubated at 4°C overnight. The homogenates were
Materials and methods
75
taken in a separate centrifuge tube and centrifuged at 7500 g for 15 min in a
refrigerated centrifuge at 4°C and the supernatant solutions were collected.
Electrophoresis
Polyacrylamide Gel (PAG) is a synthetic gel. This can be prepared from pure
acrylamide monomers and cross linkers and hence free from any contaminant. PAG
is thermo stable, transparent, strong and relatively inert chemically. PAG can be
prepared with a wide range of average pore sizes as well as gradient gels. PAG is
non-ionic and hence exhibits least Electroendosmosis. PAG can withstand high
voltage gradients, buffers with extreme pH, detergents and other chemicals and
reagents used in electrophoresis. PAG is feasible to various staining and destaining
procedures and can be digested to extract separated fractions or dried for
autoradiography and permanent recording.
Acrylamide is a white crystalline powder that forms the major ingredient in
polyacrylamide gel. As it dissolves in water “autopolymerization” takes place. It is
a slow spontaneous process by which acrylamide molecules join together by head
on tail. But in the presence of a free radical generating system, acyrlamide
monomers are activated in to a “free radical” state. These activated monomers
polymerize quickly and form long chain polymers. A solution of these polymer
chains becomes viscous but does not form gel because, these polymers slide over
one another. Gel formation requires hooking various chains together. This is done
by polymerizing acrylamide in the presence of a cross linker. N, N‟-
methylenebisacrylamide, commonly called as „bis‟ is the most frequently used cross
linking agent for polyacrylamide gels. Acrylamide and bis when activated by free
radicals form a mesh like gel.
Two proteins of different sizes, but identical charge densities, could be separated by
Polyacrylamide gel electrophoresis (PAGE) since the molecular sieving effect would
slow down the migration rate of the larger protein relative to that of the smaller
protein.
Materials and methods
76
PAGE is carried out as Native PAGE and Sodium dodecyl sulphate (SDS) PAGE. In
native PAGE, separation takes place according to both size and charge difference of
molecules. In SDS–PAGE, proteins are denatured by heating in the presence of
excess SDS and a thiol reagent (usually 2-mercaptoethanol). During this treatment
most polypeptides bind SDS in a constant weight ratio such that they have
essentially identical charge densities and migrate in polyacrylamide gels of the
correct porosity according to polypeptide size.
Principle
Acrylamide gels are formed by polymerizing acrylamide with a ‘bis’ in the
presence of a catalyst TEMED. Polymerization is accelerated by addition of
ammonium per sulphate with free radical source. The rate at which the gels
polymerise can be controlled by varying the concentrations of TEMED and
ammonium persulphate. The relative proportions of acrylamide monomer and ‘bis’
can control the porosity of the gel.
Sodium dodecyl sulphate is the common dissociating agent used to denature native
proteins to individual polypeptides. When a protein mixture is heated to 100˚C in
presence of excess SDS, the detergent wraps around the polypeptide backbone. It
binds to polypeptides in a constant ratio of 1.4g/g of polypeptide. SDS binding also
imparts a large negative charge and shadows any other charge previously present
on the polypeptide. Thus, polypeptides after treatment become a rod like structure
possessing a uniform charge density, namely same net negative charge per unit.
The mobilities of the proteins are a linear function of the logarithms of their
molecular weights.
Preparation of solutions
i) Acrylamide- bisacrylamide solution
Acrylamide - 30g
Bisacrylamide - 0.8g
Materials and methods
77
Both these were dissolved in 80mL distilled water and made upto 100mL,
filtered and stored at 4ºC in a brown bottle.
ii) 1.5M Tris-HCl, pH 8.8
Tris base -18.15g
It was dissolved in 80mL distilled water, pH adjusted to 8.8 with 1N HCl and
made up to 100mL and stored at 4ºC.
iii) 1.5M Tris-HCl, pH 6.8
Tris base -6.05g
It was dissolved in 60mL distilled water, pH adjusted to 6.8 with 1N HCl and
made up to 100mL and stored at 4ºC.
iv) 1N HCl solution
10mL concentrated HCl was mixed with 120mL distilled water.
v) 10% Ammonium persulphate (APS)
APS (100mg) was dissolved in 1mL distilled water (freshly prepared).
vi) 10% Sodium dodecyl sulphate (SDS)
SDS (1g) was dissolved in little distilled water, and made up to 10mL with
distilled water and stored at room temperature.
vii) 0.5% Bromophenol blue - 0.05g of bromophenol blue in 10mL distilled water
viii) 5X Sample Buffer
Distilled water – 2.6mL
0.5M Tris Hcl pH 6.8 – 1mL
Mercaptoethanol – 0.8mL
Glycerol – 1.6mL
10% SDS – 1.6mL
0.5% bromophenol blue – 0.4mL
Materials and methods
78
Total volume – 8mL and it was stored at room temperature.
ix) Electrode Buffer
Glycine - 4.32g
Tris base - 0.9g
SDS - 0.3g
Distilled water - 300mL
x) Staining solution
Coomassie brilliant blue R-250 - 0.1g
Methanol - 40mL
Acetic acid - 10mL
Distilled water - 50mL
xi) Destaining solution
Methanol - 40mL
Acetic acid - 10mL
Distilled water - 50mL
xii) Separating gel composition
Acrylamide - 3mL
Distilled water - 2.5mL
Tris Hcl pH 8.8 - 1.875mL
10% SDS - 75µL
10% APS - 40µL
TEMED - 5µL
Total - 7.5mL
xiii) Stacking gel composition
Acrylamide - 0.65mL
Materials and methods
79
Distilled water - 3.25mL
Tris Hcl pH 8.8 - 1.25mL
10% SDS - 50µL
10% APS - 25µL
TEMED - 5µL
Total - 5mL
Procedure
SDS-PAGE was performed following the method of Laemmli (1970) using a
separating gel of 12% and a stacking gel of 4% to separate the plant protein. The
samples containing equal amount of proteins were loaded into the wells of
polyacrylamide gels (Sigma-Aldrich Techware system; Sigma). Broad range, pre
stained molecular weight marker (Bio-Rad Laboratories, Hercules, CA) mixed with
sample buffer was also loaded in one of the wells. Electrophoresis was carried out
at constant voltage of 75 volts. Gels were stained with 0.2% Coomassie brilliant blue
R250 solution overnight and then destained. Based on the Rf value of each protein
band stained, the molecular weight was calculated.
Ammonium Sulphate Fractionation of Proteins
The solubility of proteins is markedly affected by the ionic strength of the medium.
As the ionic strength is increased, protein solubility at first increases. This is
referred to as salting in. However, beyond a certain point the solubility begins to
decrease and this is known as salting out.
At low ionic strengths the activity coefficients of the ionizable groups of the
proteins decreases so that their effective concentration decreases. This is because the
ionisable groups become surrounded by counter ions which prevent interaction
between the ionisable groups. Thus protein – protein interactions decreases and the
solubility increases.
Materials and methods
80
At high ionic strengths much water becomes bound by the added ions and remains
to properly hydrate the proteins. As a result, protein–protein interactions exceed
protein–water interactions and the solubility decreases.
Because of the differences in structure and amino acid sequence, proteins differ in
their salting in and salting out behavior. This forms the basis for the fractional
precipitation of proteins by means of a salt.
Ammonium sulphate is a particularly useful salt for the fractional precipitation of
proteins. It is available in highly purified form, has great solubility allowing for
significant changes in the ionic strength and is inexpensive. Changes in the
ammonium sulphate concentration of a solution can be brought about either by
adding solid substance or by adding a solution of known saturation, generally, a
fully saturated (100%) solution. Final concentration of ammonium sulphate used in
the present study is given in the Table A .
During 20% of protein precipitation from the sample, the initial concentration of the
sample was 0%, to precipitate the proteins from the sample, 10.7g of ammonium
sulphate was added. During 40% of protein precipitation, the initial concentration
of the sample was 20% to precipitate the proteins from the sample 11.5g of
ammonium sulphate was added. For precipitating 60% of proteins from 40%
concentrate sample 12.2g of ammonium sulphate was added. Similarly for
precipitating 80% proteins from 60% and 100% proteins from 80%, 13.1g and 14.1g
of ammonium sulphate were added respectively. For precipitation of each
concentration, the ammonium sulphate was added in small amounts by placing the
sample beaker in a magnetic stirrer by constant stirring and the sample was left for
overnight stirring in cold condition.
Materials and methods
81
Table A: Final concentration of ammonium sulphate, % saturation at 0˚C
20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
10.7
8.0
5.4
2.6
0
13.6
10.9
8.2
5.5
2.7
0
16.6
13.9
11.1
8.3
5.6
2.7
0
19.7
16.8
14.1
11.3
8.4
5.7
2.8
0
22.9
20.0
17.1
14.3
11.5
8.5
5.7
2.8
0
26.2
23.2
20.3
17.4
14.5
11.7
8.7
5.8
2.9
0
29.5
26.6
23.6
20.7
17.7
14.8
11.9
8.8
5.9
2.9
0
33.1
30.0
27.0
24.0
21.0
18.2
15.0
12.0
9.0
6.0
3.0
0
36.6
33.6
30.5
27.5
24.4
21.4
18.4
15.3
12.2
9.1
6.1
3.0
0
40.4
37.3
34.2
31.0
28.0
24.8
21.7
18.7
15.5
12.5
9.3
6.2
3.1
0
44.2
41.1
37.9
34.8
31.6
28.4
25.3
22.1
19.0
15.8
12.7
9.4
6.3
3.1
0
48.3
45.0
41.8
38.6
35.4
32.1
28.9
25.8
22.5
19.3
16.1
12.9
9.6
6.4
3.2
0
52.3
49.1
45.8
42.6
39.2
36.0
32.8
29.5
26.2
22.9
19.7
16.3
13.1
9.8
6.6
3.2
0
56.7
53.3
50.0
46.6
43.3
40.1
36.7
33.4
30.0
26.7
23.3
20.0
16.6
13.4
10.0
6.7
3.3
0
61.1
57.8
54.5
51.0
47.6
44.2
40.8
37.4
34.0
30.6
27.2
23.8
20.4
17.0
13.6
10.2
6.8
3.4
0
65.9
62.4
58.9
55.5
51.9
48.5
45.1
41.6
38.1
34.7
31.2
27.7
24.2
20.8
17.3
13.9
10.4
6.9
3.4
0
70.7
67.1
63.6
60.0
56.5
52.9
49.5
45.9
42.4
38.8
35.3
31.7
28.3
24.7
21.2
17.6
14.1
10.6
7.1
3.5
0
Materials and methods
82
Dialysis
Dialysis is a process by which small molecules are selectively removed from a
sample containing mixture of both small and large molecules. Dialysis is effectively
accomplished using a special type of membrane known as semipermeable
membrane. The semipermeable membrane allows small molecules to pass freely
through, holding the large molecules inside. These membranes are essentially made
up of cellulose derivatives.
Pretreatment of dialysis membrane
For biological work, the membranes are pretreated to remove some undesirable
impurities such as glycerol, heavy metals, sulphides, etc., that are associated during
manufacturing process. To remove glycerol, heavy metals, sulphur and also to
inactivate any enzyme that may be present in the dialysis tube, the tube is cut into
pieces of about 5-10cm, placed inside a beaker containing 500mL of 2% sodium
bicarbonate, 10mM EDTA and boiled for 5 min. The membrane is boiled in distilled
water for 5 min., the procedure is repeated again with sodium bicarbonate solution
and then with water and stored in 20% methanol at 4ºC.
Dialyzing the protein sample
The protein sample was dialyzed in the membrane by tying one end with a thread
and the samples were filled in through the other end. The tubing was checked for
leakage. The dialysis bag was placed in the phosphate buffer solution and dialyzed
overnight on a magnetic stirrer. The temperature was maintained at 4ºC throughout
the process. The proteins were collected in a centrifuge tube and stored at 4ºC the
next day.
Estimation of proteins
The protein in a solution can be measured quantitatively by different methods. The
method described by Bradford uses the concept, namely the protein‟s capacity to
Materials and methods
83
bind a dye, quantitatively. This method is simple, rapid and inexpensive (Jagadish
et al., 2010; Salekdeh et al., 2002).
Principle
The assay is based on the ability of proteins to bind coomassie brilliant blue G250
and form a complex whose extinction coefficient is much greater than that of the
free dye.
Reagent preparation
Dye concentrate
Coomassie brilliant blue G250 (100g) was dissolved in 50mL of 95% ethanol.
Concentrated ortho phosphoric acid (100mL) was added followed by
distilled water to a final volume of 200mL. The solution was stored in amber
bottles and refrigerated.
One volume of the concentrated dye solution was mixed with 4 volumes of
distilled water.
Procedure
A series of protein samples in the test tubes in the concentration 0.2, 0.4, 0.6, 0.8 and
1mL was prepared in phosphate saline buffer. The experimental samples were
prepared in 100µL of phosphate buffer saline. Diluted dye binding solution of 5mL
was added to each tube. The tubes were mixed well and left aside for 5 min for the
colour to develop but not more than 30 min. The red dye turned blue when it binds
with protein. The absorbance was read at 595nm. A standard curve was plotted
using the standard protein absorbance Vs concentration. The protein concentration
was calculated using the standard curve.
Materials and methods
84
Protein purification
Ultrafiltration
This is a simple method of purification. Various molecular cut off units like 3kDa,
10kDa and 30kDa were used and the proteins above 30kDa molecular weight were
collected and stored at 4ºC.
Reverse phase high pressure liquid chromatography (RP-HPLC)
The protein sample of more than 30kDa were analysed using RP-HPLC for
separating the protein as fractions. A shimadzu LC20AT system equipped with a
quarternary pump and a fluorescence detector was used. An isocratic elution was
performed for 70 min using the stationary phase of Hibar C18 column (250x4.6mm
i.d., 5µ) and a mobile phase consisting of buffer A- 0.1% trifluoroacetic acid (TFA) in
water and buffer B-0.1% TFA in acetonitrile (ACN). Fractions were collected in
every 5 min. the 1st fraction was collected at 0-5 min, 2nd at 6-10 min, 3rd at 11-15
min, 4th at 16-20 min, 5th at 21-25 min, 6th at 26-30 min, 7th at 31-35 min, 8th at 36-40
min, 9th at 41-45 min, 10th at 46-50 min, 11th at 51-55 min, 12th at 56-60 min, 13th at 61-
65 min and 14th fraction at 65-70 min. All the 14 fraction collected were concentrated
in a concentrator and then screened for in vitro anticancer activity.
4.9 In vitro anticancer studies
In vitro short term toxicity studies
The short term toxicity studies were carried out against Dalton‟s Lymphoma
Ascities (DLA) cells using standard procedures (Unnikrishnan and Ramadasan
Kuttan., 1988). This test relies on a breakdown of membrane integrity determined
by the uptake of a dye such as (Tryphan blue, erythorisin and nigrosin) to which
the cell is normally impermeable.
Procedure
DLA cells were cultured in peritoneal cavity of mice by injecting intraperitoneally a
suspension of DLA cells (1.0 x 105 cells/mL). The peritoneal fluid containing cells
Materials and methods
85
were withdrawn from the peritoneal cavity of the mice between 12- 15 days with
the help of a sterile syringe. The cells were washed with Hanks balanced salt
solution (HBSS) and centrifuged for 10-15 min at 1,200 rpm. The procedure was
repeated thrice. The cells were then suspended in known quantity of HBSS and the
cell count was adjusted to 2x106 cells/mL. The diluted cell suspension was
distributed into Eppendorf tubes (0.1 mL containing 2x106 cells). The cells were
exposed to the protein sample and incubated at 37ºC for 3h. After 3h, an equal
quantity of the protein treated cells and tryphan blue (0.4%) were mixed and left for
a min and loaded into a haemocytometer. The viable and non-viable count were
recorded within two min. Viable cells do not take up colour, whereas dead cells
take up colour. The percentage growth inhibition was calculated and CTC50 value
was generated from the dose-response curves for the cell line.
Total Cells – Dead Cells % Growth Inhibition = 100 – X 100
Total Cells
In vitro cytotoxicity studies
Drug development programmes involve pre–clinical screening of a vast number of
chemicals for their specific and non–specific cytotoxicity against many types of
cells. Use of in vitro assay systems for the screening of potential anticancer agents
has been a common practice almost since the beginning of cancer chemotherapy in
1946. The National Cancer Institute now routinely measures the growth inhibitory
properties of every compound under test against a panel of 60 human tumor cell
lines which are representative of major human tumor types. There are a number of
advantages in in vitro testing using cell cultures which include analysis of species
specificity, feasibility of using only small amounts of test substances, and facility to
do mechanistic studies. A novel anticancer drug should possess cytotoxicity at low
concentration against cancerous cell lines and should be safe against normal cell
Materials and methods
86
lines even at higher concentrations (Masters, 2000). All the proteins isolated were
tested for cytotoxicity by MTT assay.
Chemicals and reagents
Chemicals such as sodium bicarbonate, sodium pyruate, L-glutamine, glucose,
HEPES, penicillin and streptomycin were purchased from Sigma, USA. Likewise,
media ingredients such as Dulbecco‟s MEM, Eagle‟s MEM Medium (Sigma, USA),
and Fetal Bovine serum were procured from GIBCO USA.
Preparation of test solutions
The protein was weighed, dissolved in distilled dimethyl sulphoxide (DMSO) and
the volume was made up to 10mL with MEM/DMEM, at pH 7.4 and supplemented
with 2% inactivated FBS/NBCS (maintenance medium) to obtain a stock solution of
1mg/mL concentration, sterilized by filtration and stored at -20˚C till use. Serial
two fold dilution of the extract was prepared from the stock solution to obtain
lower concentrations.
Cell culture maintenance
Cell lines used in this study such as HBL 100 (Normal Cell line), HeLa (Cervical
Cancer), A549 (Lung carcinoma), Hep2 (Larynx Carcinoma) and OAW42 (Ovary
cancer Cells) were purchased from the National Centre for Cell Sciences (NCCS),
Pune, India. The above cell lines were maintained in Dulbecco's Modified Eagles
medium supplemented with 2mM L-glutamine and Earle‟s BSS adjusted to contain
1.5g/L sodium bicarbonate, 0.1mM non-essential amino acids and 1.0mM of
sodium pyruvate. Penicillin and streptomycin (100 IU/100µg) were adjusted to
1mL /L. Cells were maintained at 370C with 5% CO2 atmosphere.
Cell viability
Cell viability was assessed by trypan blue dye exclusion test as reported by
Chakraborty et al., (2004). The numbers of stained and unstained cells were counted
using a haemocytometer (Improved Neubauer Brightline, USA).
Materials and methods
87
Determination of mitochondrial synthesis by MTT assay
The ability of the cells to survive a toxic insult has been the basis of most
cytotoxicity assays. This assay is based on the assumption that dead cells or their
products do not reduce tetrazolium salt. The assay depends both on the number of
cells present and on the mitochondrial activity per cell. The cleavage of tetrazolium
salt, 3-(4, 5 dimethyl thiazole-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), to a
blue formazan derivative by living cells is clearly a very effective principle on
which the assay is based. The principle involved is the cleavage of MTT into a blue
coloured product (formazan) by mitochondrial enzyme succinate dehydrogenase.
The number of cells is known to be proportional to the extent of formazan
production by the cells used (Francis and Rita, 1986).
Procedure
The cell suspensions were dispensed (100µL) in triplicate into 96-well culture plates
at optimized concentrations of 1.5 x 105 cells/mL for all the cancer cells, after a 48h
recovery period, to form formazone crystal reaction. To each well of the 96 well
microtitre plate, 0.1mL of the diluted cell suspension (approximately 10,000 cells)
was added. After 24h, when a partial monolayer was formed, the supernatant was
flicked off, and the monolayer washed once with medium. Different protein
samples (100μL each) were added to the cells in microtitre plates. The plates were
then incubated at 37ºC for 3 days in 5% CO2 atmosphere, microscopic examination
was carried out and observations were noted every 24h. After 72h, the sample
solutions in the wells were discarded and 50µL of MTT in MEM – PR (Minimum
essential medium without phenol red) was added to each well. The plates were
gently shaken and incubated for 3h at 37ºC in 5% CO2 atmosphere. The supernatant
was removed, 50µL of propanol was added and the plates were gently shaken to
solubilize the formed formazan. The absorbance was measured using a ELISA
multiwell plate reader (Thermo Multiskan EX, USA) at the wavelength of 620 nm.
The percentage growth inhibition was calculated using the following formula and
Materials and methods
88
IC50 (concentration of drug or test sample needed to inhibit cell growth by 50%)
values were generated from the dose-response curves for each cell line. The relative
viability of the treated cells as compared to the control cells was expressed as the %
cytoviability, using the following formula (Sukirtha et al., 2011);
OD value of experimental samples Percentage of viability = X 100
OD value of experimental control (untreated)
IC50 was then determined by the corresponding dose response curve.
4.9 Protein characterisation
Protein characterisation is a broad term which involves creating a profile or
fingerprint of the protein molecule‟s physical, chemical and biological properties.
Mass Spectrometry
The protein fraction was run on the SDS-PAGE gel. The protein spot was excised
from preparative gel (stained with Coomassie brilliant blue) (Salekdeh et al., 2002)
and the mass spectrometry and peptide mass finger printing and MS-MS of the
protein was done. The data from matrix-assisted laser desorption/ionization- time
of flight (MALDI-TOF) MS were searched against the data bases with MASCOT
(www.matrixscience.com) and Profound software
(http://bioinformatics.genomicsolutions.com) to identify the annotated functions
of the proteins sequenced.
Protein digestion (In gel trypsin digestion)
The excised stained gel piece (minced into 1mm3 pieces) was transferred into a
sterile microcentrifuge tube. The gel was washed with 500µL of wash solution (50%
acetonitrile, 50mM ammonium bicarbonate) and incubated at room temperature for
15 min with gentle agitation (vortex mixer on lowest setting). The solution was
removed with a pipette. The gel was washed several times with 500µL of wash
Materials and methods
89
solution (15 min each) until the Coomassie dye was completely removed. The gel
was dehydrated in 100% acetonitrile for 5 min. When dehydrated, the gel pieces
had an opaque white color and were significantly smaller in size. Acetonitrile was
removed with a pipette and the gel was dried completely at room temperature for
10-20 min in a centrifugal evaporator. The gel piece was rehydrated in 150µL
reduction solution (10mM DTT, 100mM ammonium bicarbonate) for 30 min at 56ºC.
The reduction solution was discarded with a pipette and added 100µL alkylation
solution (50mM iodoacetamide, 100mM ammonium bicarbonate) and incubated for
30 min in dark at room temperature. Again the alkylation solution was discarded
with a pipette and added 500µL of wash solution and incubated at room
temperature for 15 min with gentle agitation. The wash solution was removed and
the gel was dehydrated using 100µL 100% acetonitrile for 5 min. Acetonitrile was
completely discarded and the gel was dried at room temperature in a centrifugal
evaporator. While the gel was drying protease digestion solution was prepared.
Typically, this is a modified sequencing grade trypsin (Product number V5111,
Promega, Madison, WI). The lyophilized trypsin (20µg/vial) was resuspended in a
solution containing 50mM ammonium bicarbonate (1mL), sample (50µL) and stored
at -70ºC. The gel was rehydrated with a minimal volume of protease digestion
solution. Small gel plugs (20µL) were used. The gel piece was hydrated throughout
the digest. The gel was digested overnight at 37ºC. The samples were centrifuged
and the supernatant was transferred into a sterile tube containing tryptic peptides.
The extracted solution 50µL (60% acetonitrile, 0.1% TFA) was then added to the gel
piece and sonicated in ultrasonic waterbath for 10 min. The pooled extracted
peptides were dried by centrifugal evaporation to near dryness. Resuspension
solution 5µL (50% acetonitrile, 0.1% TFA) was added to each tube and sonicated in
water bath and gently agitated on a vortex at the lowest setting. The samples were
spinned down and spotted 0.5µL on MALDI plate followed by 0.5µL of alpha-
cyano-4-hydroxycinnamic acid matrix (10mg/mL in 50% acetonitrile, 0.1%
Materials and methods
90
TFA).The spots were dried completely and the plate was loaded into Voyager and
calibrated using internal tryptic peaks of 842.5 and 2211.1 Daltons (Da).
Protein identification
The protein identification was performed automatically by searching (NCBInr)
using MASCOT search engine (Matrix Science, UK). The following are the
paramaters used;
Type of search
Enzyme used
Fixed modification
Variable modification
Peptide and fragment mass tolerance
Mass values
Protein mass
4.10 In silico analysis
Target identification
A number of methods for the computational prediction of protein structure from its
sequence have been developed. The first requirement in the construction of the
protein structure model is the multiple sequence alignment between the templates
and the target sequence. The sequence alignment is based on identifying
structurally conserved regions (SCR) common to template and target sequences.
The multiple sequence alignment was performed using the software Discovery
Studio 3.5. Query and the template sequences were given as input.
Homology modeling
Homology modeling relies on the identification of one or more known protein
structures likely to resemble the structure of the query sequence, and on the
production of an alignment that maps residues in the query sequence to residues in
the template sequence. Homology modeling can produce high-quality structural
Materials and methods
91
models when the target and template are closely related, which has inspired the
formation of a structural genomics consortium dedicated to the production of
representative experimental structures for all classes of protein folds. Homology
modeling, also known as comparative modeling of protein, refers to constructing an
atomic-resolution model of the target protein from its amino acid sequence and an
experimental three-dimensional structure of a related homologous protein. Using
the output of the multiple sequence alignment homology modeling was carried out.
The homology modeling of the protein identified by MASCOT search was done
using the software Discovery Studio 3.5
Model validation
Model validation was carried out using SAVS server. To verify the protein model,
the co-ordinates of the protein model were submitted to PROCHECK. The stereo
chemical quality of the protein structures was examined by Ramachandran plot.
The number of residues that are in the allowed and disallowed regions of
Ramachandran plot determines the quality of the model, which indicates the region
of possible angle formation.
Gene identification of the active protein
From the obtained protein sequence, the gene was identified by BLAST search in
NCBI website, derived by automated computational analysis using gene prediction
method: GNOMON. The whole genomic sequence for this reference sequence
record is from whole-genome assembly released by the US DOE Joint Genome
Institute (JGI-PGF) and the International Brachypodium Initiative as v1.0 in
December 2009 (http://www.brachypodium.org/).