PROTEOMIC REPORTFUNDAMENTAL MEDICAL SCIENCE 1

NOORGIANI LESTARI

B1-1

Faculty of Medicine Universitas Pelita HarapanMochtar Riady Institute for NanotechnologyUniversitas Pelita HarapanFaculty of Medicine\ABSTRAKEvery human have thousands protein in different type with different function,most of all the function of living organism is controlled by protein. Protein is a polymer of amino acid that have been translated from mRNA, which was originally transcript from our DNA. We learn of proteins in order to provide a comprehensive view of the structure, function, and regulation of biological systems is called proteomics. However, the aims of the study are to determine concentration of our original sample, the distribution of proteins among fractions, determine the antigen AFP concentration in our samples serum, and measure blood glucose level whether it is normal or not. First experiment, the bradford test was done for analyzing the concentration of protein. Bovine serum albumin standart was drawn and mixed with dye regent. Then the spectophotometer was set to 595 nm and absorbance of standart and sample was measured. The result of the equation from the graph of absorbance and concentrartion is y = 1.0861x-0,2222. The second experiment was protein separation through SDS-page and there are 3 step the fractionatiom of serum protein by ethanol precipitation, preparation of ample SDS-page and stining-destaining gel. The result was stained with coomassie blue in wobble table until it was clear. Then the sample was placedon a ultraviolet transilluminator to see photo of the migration and molecular size of protein. The equation that was got y = -1,5442x+5,2166. Elisa was the third experiment, In detection of AFP antigen in our samples by using elisa. Elisa is another test to detect AFP antigen in our samples. It involves an enzyme and immunologic molecules (antibody or antigen). the antigen AFP concentration is Y=1.962x-12.72.The concentration is The last experiment is about colorimetric determination of blood sugar level. Using o-toluidine,the result is y = 262,23x-2.5685. the glucose will then binds to the o-toluidine resulting a blue-green colored that can be measured at 630nm. For conclude, the result of this study showed the normal of serum protein in the blood. Protein in our blood can be analyzed and glucose level can be known using distinctive method. The total serum proteins concentration and molecular weight of certain protein also exixtance of antigen properties in our serum can be known.

TABLE OF CONTENT

COVER.............................................................................................1ABSTRACT.2

1. INTRODUCTION4

2. MATERIAL AND METHOD..10

3. RESULT.14

4. DISCUSSION20

5. REFERENCES.23

INTRODUCTIONAmino acids is monomeric unit that make protein, and primary product that make protein in apart. There are 20 amino acids that ussually use in synthesis protein. In prespektif of chemistry amino acid chain have many variant. The position of amino acid chain show their characteristic of each other.(basic medical biochemistry:clinical approach 1996,williams and wilkins) Protein is a polymer of amino acids that join together by peptide bonds. This amino acids are very crucial in our body since it contains in every cells in our body. Protein builds up, maintains, and replaces the tissues in the body. Muscles, organs, and immune system are made up mostly of protein. Body uses the protein to make lots of specialized protein molecules that have specific jobs. For instance, body uses protein to make hemoglobin, the part of red blood cells that carries oxygen to every part of your body. Other proteins are used to build cardiac muscle. In fact, whether you're running or just hanging out, protein is doing important work like moving your legs, moving your lungs, and protecting you from disease.The body itself can make the protein, called non-essential amino acids. But, in fact, there are some amino acids that the body could not produce but it still needed in the body, referred as essential amino acids. These amino acids come from our food. It can be in animal sources (meat, egg, fish, etc), plant sources (tofu, peas, green beans, etc), or even in grain products. For your information, proteins from animal sources are a complete protein, which contains all of the essential amino acids. On the other hand, protein from plant sources are only contains several essential amino acids, therefore we refer it as incomplete protein.Figure 1. Sources of Protein.

There are four structures of protein: a) primary structure, which peptide consist of defined sequences of amino acids, b) secondary structure, is a local structure which is typically recognized by specific backbone Torsion Angles and specific mainchain Hydrogen Bond pairings. Peptides can fold or align themselves in such manner that certain patterns repeat themselves, c) tertiary structure, is the folding of the total chain, the combination of the elements of secondary structure linked by turns and loops, its stability is determined by non-bonding interactions and the disulfide bond, d) quaternary structure, which is the combination of two or more chains, to form a complete unit. (http://www.bmb.uga.edu/wampler/tutorial/).

When blood is taken and being centrifuged, the blood will separate into three parts, which are plasma, buffy coat, and erythrocytes. Plasma contains of serum and fibrinogen. However, serum itself contains of protein and water. In human blood, there are two major groups of protein in the blood are albumin and globulin that made up of up to 96% of total serum proteins. First, human serum albumin consists of a single polypeptide chain of 584 aminoacids, stabilized by 17 disulfide bridges. Human serum albumin is the most common protein in serum, it is produced in the liver and the concentration in serum is 35-50 mg/mL. It is often used as a carrier molecule because of its binding capacity, and thus mainly functions as the regulator of the colloidal osmotic pressure of the blood. The molecular weight is 67.5 kDa. (http://www.assaydesigns.com/ccpa1002-3745-3747-albumin--human-serum-28hsa 29-monoclonal-antibody.htm). Second, Human Immune Serum Globulin (HISG) is used intermittently to prevent a specific infection in normal subjects, and it is continuously to prevent recurrent infections in immunocompromised subjects. Two types of HISG preparations are available: standard human immune serum globulin for general use and special human immune serum globulin with a known antibody content for specific illnesses. In addition, some animal sera and antitoxins are still used for certain infections (eg, diphtheria), poisonings (eg, snake bites or botulism), or immunosuppression (eg, antilymphocyte globulin). Recently, human immune se-rum globulin for intravenous use has been licensed. The immunoglobulins or "-globulins" are the proteins of the plasma and tissue made in lymphoreticular tissues that have antibody activity. Although there are six classes of immunoglobulinIgG, IgM, IgA, IgD, IgE, and secretory IgAonly IgG is present in significant quantities in HISG. IgG is a glycoprotein with a molecular weight of 150,000 distributed equally between the serum and the tissues. The IgG molecule is Y-shaped with two combining sites, one at the end of each arm. (http://pedsinreview.aappublications. org/cgi/content/abstract/4/5/135).The Bradford assay is a very popular protein assay because it is simple, rapid, inexpensive, and sensitive. It works by the action of coomassie brilliant blue G-250 dye (CBBG). This dye specifically binds to proteins at arginine, tryptophan, tyrosine, histidine and phenylalanine residues. This method depends on quantitating the binding of a dye to the an unknown protein and comparing this binding to that different amount of a standard protein which usually Bovine Serum Albumin (BSA). It should be noted that the assay primarily responds to arginine residues (eight times as much as the other listed residues) so if we have an arginine rich protein, we may need to find a standard that is arginine rich as well. CBBG binds to these residues in the anionic form, which has an absorbance maximum at 595 nm (blue). The free dye in solution is in the cationic form, which has an absorbance maximum at 470 nm (red). The assay is monitored at 595 nm in a spectrophotometer, and thus measures the CBBG complex with the protein. (http://www-class.unl.edu/biochem/protein_ assay/bradford_assay.htm). However, It is monitored at 595 nm because when the dye binds to protein, the protein is absorbed maximal, and also note that it must be incubated not more than 1 hour because absorbance will increase overtime. Figure 1: Chemical Structure of Coommassie Brilliant Blue G-250Electrophoresis is the migration of charged molecules in solution in response to an electric field, and a very common method for separating protein according to their electrophoretic mobility by electrophoresis uses a discontinuous polyacrylamide gel as a support medium and Sodium Dodecyl Sulfate (SDS) to denature the protein, called Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). It is an anionic detergent which denatures proteins by "wrapping around" the polypeptide backbone and SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length, example: the denatured polypeptides become "rods" of negative charge cloud with equal charge or charge densities per unit length. It is usually necessary to reduce disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size. In separating and stacking gel, we use TEMED (tetramethylethylenediamine) which its function as a catalyst in the presence of free radical initiatiors to accelerate the copolymerization of acrylamide and diacetone acrylamide into PAD. In denaturing SDS-PAGE separations therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight.

Determination of Molecular Weight is done by SDS-PAGE of proteins of known molecular weight along with the protein or nucleic acid to be characterised. A linear relationship exists between the logarithm of the molecular weight of an SDS-denatured polypeptide, or native nucleic acid, and its Rf. The Rf is calculated as the ratio of the distance migrated by the molecule to that migrated by a marker dye-front. A simple way of determining relative molecular weight by electrophoresis (Mr) is to plot a standard curve of distance migrated vs. log10MW for known samples, and read off the logMr of the sample after measuring distance migrated on the same gel. (http://www.mcb.uct.ac.za/sdspage.htm).On the other hand, there are many methods to isolate a single type of protein from a complex mixture. Separation of one protein from all others is typically the most laborious aspect of protein purification. One of the method is protein precipitation by using ethanol or ethanol-protein precipitation or solvent precipitation. When large amounts of a water-miscible solvent such as ethanol or acetone are added to a protein solution, protein precipitate out. The conventional wisdom is that this is due to decrease of the dielectric constant, which would make interactions between charged groups on the surface of proteins stronger. However, Van Oss has found that ethanol does not decrease the dielectric constant of water much, indeed 20% EtOH at -5 has the same dielectric constant as water at 20. He finds that ethanol associates with water much more strongly than do proteins, so that its real effect is to dehydrate protein surfaces, which then associate by van der Waals forces, at least if they are isoelectric or reasonably close to it. Removal of water molecules from around charged groups would also deshield them and allow charge interactions to occur more strongly, if you have areas of opposite charge on the surfaces of two proteins. Salts tend to bind to protein surfaces and make them less isoelectric, and therefore tend to mess up ethanol precipitation, which should be carried out at low salt. www.cook.rutgers.edu

1-Wavelength selection;2-Printer button;3-Concentration factor adjustement;4-UV mode selector (Deuterium lamp);5-Readout;6-Sample compartement;7-Zero control (100% T);8-Sensitivity switch;9-ON-OFF switch.Glucose is the main source of energy used by the body and the most common carbohydrate that comes from carbohydrate foods. Glucose is classified as a mono- saccharide, an aldose, a hexose, and is a reducing sugar. It is also known as dextrose, because it is dextrorotatory (meaning that as an optical isomer is rotates plane polarized light to the right and also an origin for the D designation). It also called blood sugar as it circulates in the blood at a concentration of 65-110 mg/mL of blood. (http://www.elmhurst.edu/~chm/vchembook/543glucose.html). The blood su-gar level is the amount of glucose (sugar) in the blood. It is also known as plasma glucose level. Normally, our blood glucose levels increase slightly after we eat and insulin, produced in the pancreas, released into the blood when the amount of glucose in the blood rises. There are two major method to measure blood glucose concentration: a) enzymatic method :glucose oxidase and hexokinase, b) chemical method :oxidation-reduction method and condensation reaction (o-toluidine method) in which by colorimetric determination of BSL using the o-toluidine method. However, an instrument or device used in colometry that measures the absorbance of particular wavelength of light (400-700 nm) by specific solution is called colorimeter.

1-Wavelength selection2-Printer button3-Concentration factor adjustement4-UV mode selector (Deuterium lamp)5-Readout6-Sample compartement7-Zero control (100% T)8-Sensitivity switch9-ON-OFF switch

MATERIAL AND METHODA. Bradford TestMaterial = Bovine Serum Albumin Standard Set (0.125, 0.25, 0.5, 0.75, 1, 1.5) volume 20uL, 1 X Dye Reagent volume 1uL, cuvettes.Methods =20 uL of each concentration of standard and the unknown sample was pippeted to each microcentrifuge tubes. 1 mL of 1 X Dye Reagent was added to the tubes. Blank sample was made using a distilled water and dye reagent. The tubes then be inverted to mix the solution. Next, the tubes was incubated at room temperature for at least 5 minutes. Then absorbance was read at 595nm spectophotometricaly, by zeroing the instrument first with the blank sample.

B. Ethanol PrecipitationMaterial = microcentrifuge tube, 125uL ice cold ethanol, 250uL serum, 1mL chilled 50% ethanolMethods =Serum protein was fractionated by ethanol precipitation. To a microcentrifuge tube, 125uL ice-cold ethanol and 250uL serum was added, vortexed, and stored in ice bucket for 5minutes. Then, it was centrifuged at 7000rpm for 3minutes at 40C. then the supernantant was be decanted to another microcentrifuge label ES and was stored in the ice bucket. Using the principles of capillary action, a twisted towel paper was inserted into the microcentrifuge to draw off the last supernantant above the precipitated protein. 1mL of chilled 50% ethanol was added to wash out the last traces of supernatant. Using an automatic pipette, a repeated aspiration was performed to resuspend the pellet. Then the solution was centrifuged again at 7000rpm for 3minutes at 40C. The supernantant was decanted and a twisted towel paper was inserted to remove the last supernatant. Then the last pellet was labeled EP and stored in the ice bucket.

C. SDS PAGE sample preparationMaterial = 45uL + 50uL sample buffer, rack of boiling water, centrifuge machine, ES, and EP.Methods =Then the samples for SDS PAGE was prepared. 10uL of ES was taken out to other microcentrifuge and 45uL of sample buffer was added too. For the EP fraction, 50uL of sample buffer was added and vortexed too. Then both of the ES and EP tubes was put to a rack of boiling water bath and boiled for 5minutes. After 5minutes, the tubes was taken out, and the exterior was dried with a towel paper. The tubes then be centrifuged at room temperature for 3minutesto pellet any insoluble materials that may have precipitated during boiling. Then 5uL the samples was loaded into appropriate wells of the gel.

D. SDS PAGEMaterial = Laemmli sample buffer, TEMED, and Coomasie blue staining solution that contains 50% methanol, 0.05% coomasie brilliant blue R250, 10% acetic acid, and 40% H2OMethods =Firstly, we need to make the separating gel.1.28mL distilled water, 1.6mL 30% acrylamide, 1.04 1.5 Tris HCl Buffer (pH 8.8), 40uL 10% SDS was added in a 50mL beaker glass. Then the gel casting mould was assembled as the instruction. 40uL 10% APS solution and 4uL TEMED was added to the solution and be swirled gently for 5 seconds. Using an automatic pipette, 1.8mL of the separating gel was added to the gel casting mould. The separating gel was overlaid with distilled water immediately. Then it was left for 30minutes to let it polymerized. After polymerizing, the distilled water was poured.After the separating gel has polymerized, a stacking gel will then be casted. First, a solution containing 0.7mL distilled water, 0.165mL 30% acrylamide, 0.125mL 0.5M Tris HCl Buffer (pH 6.8), and 10uL of 10% SDS is prepared in a 50mL beaker glass. Then, 10uL 10% APS solution and 1uL TEMED was added to the solution and swirled gently for 5seconds. Using an automatic pipette, 0,5mL stacking gel was added on the top of polymerized separating gel. Then the comb was placed between the glass plates with one end higher then the other and carefully press the comb down so the teeth are level about 5mm from the top of separating gel. Then the gel was left to polymerize for 20 30 minutes. The comb was carefully removed and the wells was washed out at least 3times with distilled water.

E. Gel StainingMaterial = staining dye (coomasie brilliant blue, destain solution, wooble table, and cellophane plasticMethods =For staining the gel, the gel was removed and stained with coomasie blue in wooble table for 1hour. Then the gel was destained with a destain solution in wooble table for another 1hour and the destain solution was changed with the fresh one every 1hour until the background is clear. Then the gel was wrapped in a piece of cellophane plastic.

F. Enzyme Linked ImmunoSorbent AssayMaterial = rabbit anti human AFP coated microtiter plate with 96 wells, 13mL zero buffer, reference standard set (contain 0, 5, 20, 50, 150, nad 300ng/mL AFP, lypophilized), 18mL enzyme conjugate reagent, 11mL TMB reagent, 11mL stop solution (1N HCl), distilled water, and microtiter plate reader (with a bandwidth of 10nm or less and an optical density range of 0-2 OD or greater at 450nm wavelength).Methods =The desired number of coated wells was secured in the holder. 20uL of standard, specimens, and controls was dispensed into appropriate wells. 100uL of zero buffers was dispensed to each well and mixed for 30seconds. Then it was incubated at room temperaure ( 18 250C) for 30minutes. The incubation mixture then be removed by flicking plate content into a waste container. After that, the microtiter wells was rinsed and flicked 5times with distilled water. The wells were striked sharply onto absorbent paper to remove all residual water droplets. Next, 150uL of enzyme conjugate reagent was dispensed into each well and mixed gently for 5seconds. Then it was incubated at room temperature for 30minutes. The incubation mixture was removed by flicking plate contents into a waste container. Then the microtiter wells was rinsed and flicked 5times with distilled water. Then the wells were strike sharply onto absorbent paper to remove residual water droplets. 100uL of TMB reagent was dispensed into each wells and mixed gently for 5seconds. Then it was incubated at room temperature for 20minutes. Then 100uL of stop solution was added and mixed for 30seconds to each well to stop the reactions. Make sure that all the blue color changes to yellow color completely. Then the optical density was read at 450nm with a microtiter reader within 15minutes.

Colorimetric Determination of Blood Sugar Level Material = o toluidine reagent, standard glucose solution 12mg/dL, distilled water, spectophotometer, cuvette, reaction tube, aluminium foil, and water bath.Methods = Glucose standard is provided. Concentration of glucose standard point is provided too. Since ours is group 3, so we did the tubes no 3, which the concentration was 100 mg/dl, 1 ml glucose standard, 1 ml water, and the total per tube was 2 ml. Then, 2 ml 1% O-toluidine was added, mixed well, and incubated. After that, the tubes were put in a boiling water bath for 10 minutes. Last, measured at absorbance 630 nm.For measuring blood glucose, serum from the last genomic experiment was obtained. Then the glucose concentration was determined by using o toluidine methods. First, 0.05mL of standard and 1mL of o toluidine was added in a clean dry test tube and mixed gently. Then the tubes were put in a rack of boiling water for 10minutes. After 10minutes, the tubes were taken out and washed under tap water. The absorbance was read at lmax 630 nm. Finally, by using the absorbance reading of standard glucose, calculated the concentration of BSL in the provided blood samples.

RESULTBRADFORD TEST EXPERIMENTCONCENTRATIONAVERAGE A595

0,1250,106

0,250,219

0,50,4

0,750,582

10,746

Sample 11st test = 0,8292nd test = 0,835Absorbance(mean) = 0.829+0.835/2=0.832 the absorbance in the equation : 0,832=1.0861x+0.22221.0542=1.0861x X=0,971X(times dilution factor)=0,971 mg/mlSample 21st test = 0,8542nd test = 0,860Absorbance (mean)= 0.854+0.860/2=0.857the absorbance in the equation :0.857=1.0861x+0.22221.0792=1.0861xX=0.994X(times dilution factor)=0.994 mg/ml>> dilution factor that use in this experiment is 1 : 100 , this is due to fix into the graph range. Because of this delution factor, after the calculation from equation we need to multiply the result 100 times to get the protein concentration. after the calculation from the equation we need to multiply the result 100 times to get the protein concentration. So that the results become: 97,1 mg/ml for sample 1 and 99,4 mg/ml for sample 2.

SDS-PAGE

L = 5.1 cm0.8 cm1.6 cm2.6 cm1.9 cm Es Sample 11.2 cm Ep sample 11.2 cm Es sample 21.9 cm -Ep sample 250 kDa25 kDa

sizeDistance(cm)RF(distance/length)Log size

252.5 0.5324.39

501.6 0.3404.69

7510.2134.87

1000.70.1495

Retention factor ( Rf ) = distance protein migrated / length of the gel.Length of the gel = 4.7 cm.

The chart above was result of sds-page electrophoresis. The left one was protein marker, which range from the smallest molecular weight. We couldnt see the difference between the Ep and Es, the black dots in the middle had the largest number. And compare the protein marker with a certain protein and by calculation of the equation between Rf and log MW (Molecular Weght),the information that we can see about molecular weight was obtained. Therefore, the retention factor ( Rf ) are :size

25

50

75

100

2.5 /4,7cm=0,53

1.6 /4,7cm=0,34

1/4,7cm=0,21

0.7/4,7cm=0,14

RF :

From the DNA ledder, the curve produced has the equation y = -1,5442x+5,2166Sample supernatant :Y = - 1,5442(0,36)+5,2166Y = 4.6606Log size = 4.6606Size = 10 4.6606 = 45772 daltonSize = 45.772 kDaSample pellet :Y = - 1,5442(0,23)+5,2166Y = 4.861Log size = 4.861Size = 10 4.681= 72610 dalton Size = 72.610 kDaELISA

B1-1-1 = 0,018B1-1-2=0,014CONCENTRATION Y=1.962x-12.72B1-1-1 = 196.2(0.018)-12.72= -9.1884mg/dlB1-1-2=196.2(0.014)-12.72= -9.9732 mg/dlR2= 0.984

Colourimetric Determination of Blood Sugar LevelConcentration (mg/dL)Absorbance

600.234

800.352

1000.361

1200.465

The resul of our sample calculation :TubeAbsorbanced(nm)Absorbanced2(nm)Average(nm)

Sample 10.3510.3540.3525

sample 20.3830.3850.3840

standart0.2380.2370.2375

Sample 1 = 60 * 0.3525 / 0.2379 = 89.052 mg/dLSample 2 = 60 * 0.3840 / 0.2375 = 97.01 mg/dLDISCUSSIONIn the Bradford test, the BSA/standard protein contain approximately 1 mg/ml. But the sample protein of normal healthy adult (sample 1 and 2) contain 55-90 ml. Therefore, it needs to be diluted 100x, so that the concentration becomes less than 1 mg/ml. The absorbance can only be measure with those standard. Its discussed about two types of bonds interaction take place here, the red form coomasie dye first donates its free proton to the ionizable groups on protein,which cause a disrupture of proteins native state,and consequent expose its hydrophobic pockets. The exposed hydrophobic pockets on the protein will bind non-covalently to the non polar region of the dye via van der walls force, this will position the positive amines groups to proximity with the negative charge of dye, and the bond is further strengthened by the ionic interaction between the two. Binding of the protein stabilized the blue form of coomasie dye.In this bradford assay, we measure the absorbance with spectrophotometer at 595 nm because at this lambda, the absorbance is in the optimum point,where absorbance was at the peak and cant go up any furher.The standard curve shown that the equation is y = 1.0861x-0,2222.. From the first test of sample 1, the measurement is 0.681, and second test is 0.689, so that the average is 0.685. While from the sample 2, first test is 0.936 and second test is 0.940 thus the average become 0.938. From the calculation above the concentration of sample 1 and sample 2 are 83.224 mg/ml and 106.095 mg/ml. here we can see that the protein concentration of sample one lies between the range (50-90mg/ml) and sample 2 doesnt lie between the standard range due to excessive protein. It happen because theres an error mixed or pipetting the volume of unknown sample or dye reagent while we did the experiment. If the sample of protein drew under 20 l, it causes a decrease in number of total serum protein. If the protein concentration is higher than the normal range that means that person has an excess protein concentration. Commonly it caused by an abnormal diet which replacing the carbohydrates with the protein. This condition is dangerous because protein makes up more than 30% of your caloric intake causes a buildup of toxic ketones. So-called ketogenic diets can thrust your kidneys into overdrive in order to flush these ketones from your body. As your kidneys rid your body of these toxic ketones, you can lose a significant amount of water, which puts you at risk of dehydration, particularly if you exercise heavily. That water loss often shows up on the scale as weight loss. But along with losing water, you lose muscle mass and bone calcium. The dehydration also strains your kidneys and puts stress on your heart. And dehydration from a ketogenic diet can make you feel weak and dizzy, give you bad breath, or lead to other problems, such as colon and rectum cancer, arthritis and gout, and also kidney and liver hypertrophy. (http://www.medicinenet.com/script/main/art.asp?articlekey=50900)The normal range of the AFP antigent itself is below 20 mg/mL. Since the AFP concentration of sample is - 22.42 mg/mL and for sample, the AFP concentration is 21.74 mg/mL, so both sample has a normal concentration of AFP antigent. If the AFP concentration is higher than the normal range, it indicates hepatocellular carcinoma, germ cell (nonseminoma) carcinoma, normal pregnancy, benign liver disease (hepatitis, cirrhosis), as well as in cancer, and also testicular germ cell tumors containing embryonal or endodermal sinus elements.The AFP is rarely elevated in healthy persons, and a rise is seen in only a few disease states. Elevation occurs in certain liver diseases, especially acute viral or drug induced hepatitis and conditions associated with hepatic regeneration. In general, the elevations are under 500 ng/ml and do not denote hepatocellular carcinoma. Is also elevated in ataxia-telangiectasia and in hereditary tyrosinosis. (http://www.tc-cancer.com/tumormarkers.html. Our result shown that both sample are less than the normal range, sample 1 is -9.77ng/ml, and sample 2 is -9.9732 ng/ml, which that means they not detected to cancer or subset tumor.The normal range of the glucose concentration in blood is 70 100 mg/mL. Our result for colorimetric glucose concentration in Michael serum is89.052 mg/mL, on the other hand, for Admiral serum, the glucose concentration is 96.072 mg/mL.If our glucose concentration in blood serum is below than 70 mg/mL, it means we have a low blood sugar level. This condition is called hypoglycemia. Hypoglycemia causes symptoms such as hunger, shakiness, dizziness, sleepiness, confusion, difficulty speaking, and weakness. Hypoglycemia could commonly caused by side effects of some medications.( http://www.nlm.nih.gov/medlineplus/hypoglycemia.html).Our both sample of blood sugar level were 82.13179 mg/dl and 79.640605 mg/dl. Compared with the normal range of blood sugar level. Both of the samples are in the normal condition. The high level of the blood sugar may be indicating problems in our insulin hormone. Known as Diabetes mellitus. After all the conclusion is from all the experiment weve done we know that protein in our blood can be identify through several experiment, and it give us the information about the concentration of our protein, molecular weight of certain protein and existence of antigen properties in our serum also the blood sugar level. , if the glucose concentration is above 100 mg/mL, it indicates a condition referred as hyperglycemia, high blood sugar level. Hyperglycemia is commonly caused by diabetes, the effects of insulin on your body are drastically diminished, either because your pancreas doesn't produce enough of it (type 1 diabetes) or because your cells are less responsive to it (type 2 diabetes). As a result, glucose tends to build up in your bloodstream and may reach dangerously high levels. To correct this problem, people with diabetes take insulin or other drugs designed to lower blood sugar levels. (http://www.mayoclinic.com/health/hypoglycemia/DS00198/DSECTION=causes).

References1. (williams and wilkins)basic medical biochemistry:clinical approach 1996,2. . (http://www.bmb.uga.edu/wampler/tutorial/).3. (http://www.assaydesigns.com/ccpa1002-3745-3747-albumin--human-serum-28hsa 29-monoclonal-antibody.htm).4. . (http://pedsinreview.aappublications. org/cgi/content/abstract/4/5/135).5. (http://www-class.unl.edu/biochem/protein_assay/bradford_assay.htm).6. (http://www.mcb.uct.ac.za/sdspage.htm).7.(http://72.14.235.132/search?q=cache:pSAvTirC_wMJ: www.cook.rutgers.edu/~dbm/precipitations04.pdf+protein+precipitation+using+ethanol&hl=id&ct=clnk&cd=3&gl=id)8. (http://www.elmhurst.edu/~chm/vchembook/543glucose.html).9. (http://www.medicinenet.com/script/main/art.asp?articlekey=50900)10. http://www.tc-cancer.com/tumormarkers.html.11. ( http://www.nlm.nih.gov/medlineplus/hypoglycemia.html)12. (http://www.mayoclinic.com/health/hypoglycemia/DS00198/DSECTION=causes).

Noorgiani LestariPage 23