Post on 06-Jun-2022
Biochemistry Biochemical Techniques
21 Western Blotting
Biochemistry Biochemical Techniques
21 Western Blotting
Description of Module
Subject Name Biochemistry
Paper Name 12 Biochemical Techniques
Module Name/Title 21 Western Blotting
Biochemistry Biochemical Techniques
21 Western Blotting
1. Objectives
1.1 To understand principle of Western Blotting technique
1.2 To explain how this techniques is performed
1.3 What are helpful hints which help in successful performance of Western Blotting
Technique?
2.0 Introduction and Principle-
Electrophoretic techniques are very handy for analysis of charged particles. However,
the method lacks high degree of resolution when sample is complex and contains
molecules similar to analyte. When other scientific principles are coupled to
electrophoretic resolution, results are derived with higher degree of certainty. This is
the concept in all blotting techniques where molecules separated on gel are transferred
to membrane for their subsequent analysis. Western blot refers to transfer of protein
from gel to membrane and this technique was described in 1979-80 by many workers
but the method described by Towbin (1979) is most cited. The transfer of protein from
gel to membrane is electrophoretically achieved. The use of capillary flow to transfer
DNA from agarose gels to nitrocellulose membrane was first described by Southern
(1975) and thus referred as Southern blotting. Using the same method for transfer of
RNA to membrane is referred as Northern blotting.
The membrane materials frequently employed in blotting are nitrocellulose, nylon and
polyvinylidene difluoride (PVDF). The choice of membrane depends on the type of
analysis and characteristics of detection system. Nitrocellulose is the most widely used
since it works well with both protein and nucleic acids. Some nylons do not bind protein
reliably. PVDF is often used when bound proteins are analysed for sequencing.
Western blotting essentially comprises of three techniques which are applied in
sequence. The first one is referred as SDS-PAGE through which proteins are separated
based on the molecular size of molecules in acrylamide gel. Sodium dodecyl sulphate
(SDS) is an anionic detergent that denatures proteins by wrapping around the
polypeptide backbone. This results in net negative charge to polypeptide in proportion
to its length. Laemmli system (Laemmli, 1970) employing discontinuous buffer is most
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widely used electrophoretic system. The resolution in Laemmli's method is excellent
because treated proteins or peptides are concentrated in stacking gel before entering
the separating gel. Proteins from SDS-PAGE gel are electrophoretically transferred to
membrane. There are two types of equipments for electrophoretic transfer of proteins:
the semi-dry blotting apparatus and 'tank' buffer apparatus. The third technique used in
sequence is for identification of protein (antigen) by performing" antigen-antibody (first
antibody) reactions on the membrane itself. Second antibody enzyme conjugates were
then allowed to interact with immobilized first antibody and, then using appropriate
substrate, protein bands are detected. Although, antigen-antibody interactions are
widely employed in Western blot, other kind of interactions such as glycoprotein-lactin
and biotin-avidin have allowed research workers to employ this technique for other
applications including carbohydrate staining of glycoprotein, protein sequencing etc.
3.0 Blot Membranes
Numerous types of papers and membranes have been utilized for protein blotting.
Nitrocellulose paper (film of nitric acid esterified cellulose) has been the most frequently
used membrane. The binding of proteins to nitrocellulose is probably hydrophobic. For
electrophoretic transfer of small proteins, membranes with 0.1 or 0.2µm pore size are
selected. If membranes stick to low concentration gels' after transfer, membranes with
pore size of 0.45 µm are selected. A drawback with nitrocellulose membrane is,
however, that they are very brittle when dry.
The other membrane, which is in use, is polyvinyllidene fluoride (PVDF). PVDF
membrane is a teflon-type polymer composed of the basic repeating unit(- 8+CH2-8-CF2-
)n and has good mechanical strength. Proteins interact with the polymer non-covalently
through dipolar and hydrophobic interactions. PVDF is chemically compatible with the
aqueous buffer systems. Since PVDF is resistant to most organic solvents and it can
withstand harsh chemical conditions in which nitrocellulose membranes dissolve or
decompose. These membranes are expensive. Some PVDF membranes have additional
components. PVDF can also be casted on polyester web. The web does not interfere
with electroblotting or alter the characteristics of the PVDF. Immobilon-CD is PVDF
membrane in which surface is chemically modified to have a cationic charge. Although
hydrophobic and dipolar interactions with the Immobilon - CD may contribute to protein
binding, the primary binding interaction is ionic. The membranes with high internal
surface area (>2000cm2 per cm2 of frontal area) bind substantially more protein (400 µg
BSA I cm2) as compared to membrane with low internal surface area (~400 cm2 per cm2
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21 Western Blotting
of frontal area) that binds to around 130 µg BSA I cm2. Low internal surface area
membranes usually function better in immunodetection. They are comparatively easy to
block and antibodies are better able to penetrate large pore structure. Membranes with
high internal surface structure are more difficult to block effectively and less open pore
structure often limits antibody accessibility. Besides immunodetection, PVDF
membranes are used for amino acid sequencing, amino acid analysis and peptide
mapping. For these applications blocking is not required and there is no steric hindrance
encountered by antibodies. Higher internal surface membranes and Immobilon CD are
suitable for amino acid sequencing or amino acid analysis. Peptide mapping is more
effective on low internal surface area membranes. PVDF membrane is compatible with
protein staining and immune-chemical protocols.
Positively charged nylon membranes arc mechanically strong and have a high binding
capacity. A disadvantage is their high non-specific binding which results in a high
background after immunodetection. Most general protein stains are anionic dyes and
cannot be used with nylon membranes since they bind to these membranes.
4.0 Transfer Unit
4.1 Semi-dry Electrophoretic Transfer
In semi-dry electrophoretic transfer, a stack of wetted filter papers surrounding the gel
and the blotting membrane is used as a buffer reservoir, instead of tank as in
conventional electrophoretic transfer. The electrodes consist of conductive plates made
of graphite or stainless steel or a conducting polymer. The size of the plates is at least
the same size as that of gel to provide homogeneous electric field. The main advantages
with semi-dry transfer are the ease of handling, the short time (30 min, to 1 h) required
for the transfer and low buffer consumption. Another important feature is that different
buffers can be used at the anodic and cathodic sides to improve the transfer. The short
electrode distance gives a high voltage gradient despite low power. Cooling is not
normally required since heat production is negligible. Transfer can be performed from
several gels at a time, either by placing them beside each other if the electrodes are
large enough or by placing several transfer units on top of each other. Because of the
short electrode distance, voltage applied is most often 10-20V. On the other hand,
because of large cross-sectional area, the current passing through the transfer sandwich
is fairly high, in the range 0.1-1A.
4.2 Tank-Buffer Electrophoretic Transfer
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In tank-buffer electrophoretic transfer, transfer cassette is submerged in a 'tank' of
buffer. Gel, membrane, filter paper, porous foam sheet are placed in defined way in
cassette as per instructions of manufacturer. This is widely used method.
5.0 Transfer Buffer
A major concern in transferring proteins onto nitrocellulose membrane is the
composition of the transfer buffer. The original protocol of Towbin et al. (1979) uses a
transfer buffer containing methanol, which was added to counteract swelling of the gel.
Methanol also decreases gel pore size, removes SDS from proteins. Methanol may
precipitate the proteins within the gel, however it increases the capacity and the affinity
of nitrocellulose membrane for proteins. PVDF membrane is activated by placing it in
100% methanol for 1-2 sec. This allows the hydrophobic surface of PVDF to wet with
aqueous solvent. Addition of 20% methanol to transfer buffer is recommended for low
molecular weight proteins. Methanol is not required in transfer buffer when proteins
are transferred to charged nylon membranes. Methanol facilitates the dissociation of
SDS-Protein complexes and increases the hydrophobic interaction between protein and
membrane. On the other hand, for high molecular weight proteins, methanol can
decrease the elution efficiency by denaturing the proteins or retarding the elution from
the gel. In contrast to low molecular weight proteins, high molecular weight proteins do
not require methanol for adequate binding to the membrane.
The presence of SDS in transfer buffer increases the mobility of protein from gel to
membrane. This is especially useful for transfer of protein after isoelectric focussing,
when proteins have no net charge. However, SDS decreases the binding of the protein
to both nitrocellulose and PVDF membrane. It is sometimes necessary to add SDS (0.01-
0.02%) to aid transfer of high molecular weight proteins. Transfer buffer generally used
is 25 mM Tris, 192 mM glycine, pH 8.3 and 20% methanol. If membrane is to be used
for protein sequencing or amino acid analysis, CAPS buffer (10 mM 3-(cyclohexylamino)-
1 propanesulonic acid, 10% methanol, pH 11.0) is recommended. Application of protein
blotting in antigens characterization will require antigen specific antiserum. By
simultaneously running molecular weight markers and proteins (extracted from
biological materials) in SDS-PAGE and subsequent detection after electrophoretic
transfer provides information about molecular weight of antigen. Antibodies should be
specific otherwise cross-reaction is observed and interpretation is more difficult. Affinity
purified antibodies or monoclonal antibodies provide good result. Through these
reactions, one can detect presence or absence of such antigens in related and unrelated
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21 Western Blotting
biological materials. Now tools are available for ascertaining carbohydrate moiety in
proteins on membrane. These proteins can be oxidized by periodate resulting in
generation of free aldehyde groups. The generated groups are reacted with biocytin
hydrazide leading to biotinylation of glycoproteins. Using appropriate probe such as
avidin-peroxidase and substrate, glycoproteins are detected. Alternately lactins specific
for carbohydrate residues can be employed. In this approach antibody (against lactins)
enzyme conjugates or lactin - enzyme conjugates can be used for staining glycoproteins.
Proteins onto membrane can be hydrolyzed for determining amino acid composition.
Peptide mapping and protein sequencing are other useful applications where proteins
on membrane are the starting material for subsequent steps.
6.0 Electrophoretic Transfer of Proteins From Gel To Membrane
6.1 Equipments and Chemicals-
Mini-trans blot assembly (BioRad), power pack, orbital shaker, tris, glycine, methanol,
nitrocellulose membrane, Whatman No. 3 paper.
6.2 Transfer buffer (25 mM tris, 192 mM glycine, 20% methanol, pH 8.3)-
3.03 g tris and 14.4 g glycine are dissolved in distilled water. 200 ml methanol is then
added. Volume is made up to 1 litre with distilled water. The pH of buffer will range
from 8.1 to 8.4 depending on quality of tris, glycine and methanol. Methanol should be
analytical grade as metallic contaminants in low grade methanol will plate on the
electrode. The pH of buffer is not adjusted with acid or base.
6.3 Procedure-
Membrane and gel are handled only after wearing gloves
fter completion of SDS-PAGE run, comb and spacers are removed. Notched and
rectangular plates are gently loosened so that gel stays with rectangular plate.
Spacer gel is removed. A small cut on top left side in running gel is made to remember
the orientation of gel.
The running gel is equilibrated with transfer buffer for 30 min. to remove salts and SDS.
Transfer buffer is changed at least once during equilibration. Membrane of appropriate
size (at least size of running gel) is cut from sheet. A small cut on top left side of
membrane (glossy side facing worker) is made to remember orientation of the
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membrane. Now a days membranes having binding regions on both faces are available
and these membranes does not have glossy side.
While the gel is equilibrating, nitrocellulose membrane is placed it in transfer buffer.
Also, fiber pads and pre-cut filter papers (Whatman No. 3) are immersed in transfer
buffer. Air bubbles trapped in fiber pads and filter papers are removed. This is achieved
by immerging pads and papers in buffer and then pressed there by rolling clean-glass
test tube. At this stage gel is never allowed to be in same container containing transfer
buffer membrane, pads & papers
Gel holder cassette is opened and placed in glass vessel so that the gray panel is flat on
the bottom of the vessel and clear panel rests at an angle against wall of the vessel.
Gel holder cassette is assembled in following sequence: gray panel (cathode), fiber pad,
filter paper, gel, nitrocellulose membrane (glossy side facing the gel), filter paper, fiber
pad, clear panel (anode). For easy remembrance of orientation, cut portions of gel and
membrane is aligned. This arrangement allows transfer of proteins on membrane where
well position remains the same as that in acrylamide gel. While assembling, care is taken
not to allow trapping of air-bubbles. This is achieved by (i) assembling cassette under
buffer and (ii) when fiber pads, papers and gel are placed, all air pockets are removed by
rolling clean test tube over the layer after each placement. Nearly adhesive contact is
essential between the membrane and gel otherwise swirled or missing transfer patterns
and overall high background will be observed.
Buffer tank is filled with transfer buffer (40C). Bio-freeze cooling unit containing ice is
placed in buffer tank.
Gel holder cassette is closed and placed in the buffer tank such that gray panel of the
cassette faces the gray cathode electrode panel. The whole of blotting assembly is then
placed over the magnetic stirrer.
Electrophoretic transfer is carried out at constant voltage of 30 V overnight at 40C. The
starting current should be around 40 mA. At the end of transfer, the current should be
90 mA. In case final value of current is less than 90 mA, a constant voltage of 100 V is
additionally applied for 1 h.
After run, nitrocellulose membrane is stained with different reagents for visualization of
proteins or antigens. For ascertaining transfer of proteins from gel, the gel is also
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stained with coomassie brilliant blue and is described below.
6.4 Staining of proteins in gel-
The gel is placed in glass tray containing coomassie brilliant blue solution (0.25%)
prepared in methanol : acetic acid : water (40:7:53) mixture. Glass tray is then placed on
orbital shaker for 4 h at room temperature. After staining for 4h, the gel is transferred
to the destaining solution I (methanol, acetic acid and water mixture in ratio of (40:7:53)
for 30 min. Subsequently gel is placed in destaining solution II (methanol, acetic acid and
water mixture in ratio of 7:5:88) till bands become visible against light background.
During staining and destaining, gel should float free in glass tray.
6.5 Detection of Transferred Protein on Nitrocellulose Membrane
In Western blot, molecular weight markers and protein (antigen) samples are loaded in
separate lanes in SDS-PAGE. Whereas, methods used for staining of molecular weight
markers are based on non-specific reaction of dye with protein, antigenic proteins are
detected employing antigen-antibody interaction. Therefore, after electrophoretic
transfer, the membrane-portion containing molecular weight marker is cut from rest of
membrane containing protein antigens. It is essential to keep one lane adjacent to
molecular weight markers lane vacant to allow safe cutting of portion of membrane
containing marker lane. The molecular weight markers can be stained by Ponceau S or
congo-red dye. The proteins (antigens) are stained using primary antibody and
secondary antibody-enzyme conjugates.
6.6 Visualization of Molecular Weight Markers-
6.6.1 Ponceau S Staining-
Stock Ponceau S dye solution is prepared by dissolving 200 mg Ponceau S in 10 ml of 3%
trichloroacetic acid. The stock dye solution can be stored at room temperature. The
stock solution is diluted tenfold with distilled water before use. The membrane is added
slowly to vessel containing diluted dye solution so that membrane absorbs dye
uniformly. The membrane is then sub-merged for 5 to 10 min with mild shaking. After
staining, the membrane is then rinsed with water or PBS until a clear contrast between
the bands (pink) and background (white) is observed. Staining of proteins with Ponceau
S is reversible.
6.6.2 Congo-red Staining-
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Stock Congo-red solution is prepared by dissolving 1 g Congo-red in 100 ml distilled
water. This solution is stable at room temperature. The working congo-red solution is
prepared just before use by diluting 1 ml of stock dye solution with 9 ml of 0.2 M
acetate buffer, pH 3.5. The membrane is sub-merged in working congo-red solution for
5 min. at room temperature. The destaining is carried out by immersing the membrane
in distilled water until brown bands become visible against light pink background. During
staining and destaining, mild shaking is employed.
6.7 Visualization of Protein (Antigen)-
6.7.1 Reagents-
Primary antibodies directed against antigen and are usually raised in rabbit, secondary
antibody enzyme conjugates such as goat anti rabbit immunoglobulin-peroxidase goat,
anti rabbit immunoglobulin-alkaline phosphatase, diaminobenzidine, hydrogen peroxide,
bovine serum albumin, nitro blue tetrazolium (NBT) bromochloroindolyl phosphate (BCIP),
dimethyl formamide (DMF).
6.7.2 Visualization of antigen using secondary antibody peroxidase conjugate-
All steps are carried out at room temperature.
Membrane is washed with PBS (3x10 min.)
The membrane is treated with blocking solution (3% BSA prepared in PBS) for
1 h.
The membrane is treated with diluted rabbit antiserum for 1 h. The antiserum in rabbit
is raised against the antigen. The dilution is decided by antibody litre in immune serum
and is carried out in 1% BSA – PBS- (0.05%) Tween – 20. The membrane is washed with
PBS -.05% Tween 20 (3x10 nin.).
The membrane is treated with goat anti-rabbit immunoglobulin-peroxidase conjugate
(1:1000 diluted with 1% BSA – PBS – Tween 20) for 1 h. The dilution of conjugate is done
as per instruction from manufacturer.
The membrane is washed with PBS (4 x 10 min.).
The membrane is immersed in enzyme substrate DAB - H2O2 (6 mg diaminobenzidine in
10 ml of 0.05 M Tris-HCl buffer, pH 7.6 containing 100 µl of 3% H2O2 ) till brown bands
become visible. The membrane at that stage is washed with distilled water and air-
dried.
Membrane strips containing molecular weight markers and proteins (antigens) are
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aligned and photographed.
6.7.3Visualization of antigen using secondary antibody-alkaline phosphatase conjugate
The method is similar to the method described using secondary antibody-peroxidase conjugate except the followings.
Appropriately diluted secondary antibody-alkaline phosphatase conjugate is used instead of antibody-peroxidase conjugate.
Instead of DAB-H2O2, the enzyme substrate used is BCIP-NBT. Stock solutions of
nitroblue tetrazolium (NBT) and bromochloro indolyl phosphate (BCIP) are prepared and
stored at –200C. Stock NBT is prepared by dissolving 30 mg NBT in 1 ml of 70 per cent
DMF. Stock BCIP is prepared by dissolving 15 mg BCIP in 1 ml of DMF. The working
substrate solution is prepared by addition of 200 µl of stock NBT and 200 µl of stock
BCIP to 20 ml of 100 mM Tris-HCl, pH 9.5 containing 100 mM NaCl and 5 mM MgCl2.
When membrane is treated with enzyme substrate, light violet colour blots become
visible against light background.
7.0 Helpful-Hints-
7.1 SDS-PAGE-
A particular concentration of acrylamide gel is used for separating proteins of particular
range of molecular weights. Whereas low acrylamide gel concentration is used for
separating high molecular weight proteins, low molecular weight proteins are resolved
in high gel concentration. Use following table in deciding gel concentration in separating
gel.
______________________________________________________________ Per Cent gel Molecular weight of proteins to be separated (KD) ______________________________________________________________
7.5 24 – 205
10.0 14 – 205
12.5 14 – 66
15.0 14 – 45
--------------------------------------------------------------------------------------------
Spacers can absorb heat and thus lowers the temperature of gel at edges. If the gel is
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21 Western Blotting
hotter in the middle than at the edges, the mobility of dye front at edges will be lower
as compared to mobility in the middle. This can be avoided by (i) using cooled electrode
buffer and ii) not allowing buffer to warm up during run. Thus, during electrophoretic
run either use cooling device or use low current.
If gel is not polymerized properly at edges, current can leak down the edges resulting in
more mobility at edges. Air- bubbles at the bottom of glass plates can block current flow
resulting abnormal dye front.
While placing comb in stacking gel, care should be taken not to allow air-trap. Air
inhibits polymerization and sample wells will be distorted.
All stock solutions required for gel preparation are stored at refrigerated temperature
and these should be brought to room temperature. At low temperature, polymerization
is inhibited. Oxygen also inhibits polymerization of acrylamide and these solutions
should be degassed before use.
Sometimes boiling of sample in sample buffer may lead to irreversible precipitation and
such samples remain at the top of separating gel. For such samples one can try
incubating sample in ample buffer at 700C instead of 1000C.
7.2 Electrophoretic Transfer-
The one major problem in Western blot is incomplete transfer of protein from gel to
nitrocellulose membrane. Transfer efficiency is improved by decreasing gel concentration
which leads to more porous gel. In more porous gel, the resolution of proteins is decreased.
Gel containing low molecular weight proteins should not be excessively washed after SDS-
PAGE and before transfer to avoid removal of these proteins in washing.
Methanol in transfer improves binding of SDS-proteins to nitrocellulose membrane but
it causes acrylamide gel pores to contract resulting in fixation of large molecular weight
proteins within the gel matrix. In case of poor transfer of large molecular weight
proteins, one can try transfer in transfer buffer containing reduced concentration of
methanol.
Gel and membrane must make good contact. Thus excess moisture in the gel-
membrane interface should be removed by rolling test tube over membrane while gel
holder cassette is assembled.
Poor transfer can occur if the protein is basic (ie pI > 9) as protein will have net positive
charge at the pH of transfer buffer (pH 8.5).
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21 Western Blotting
Lower concentration of methanol (< 15%) does not facilitate removal of SDS from the
gel and proteins.
Nitrocellulose membrane is compatible with enzyme immuno assay. Blocking of free
protein binding sites is easy and thus background problems are not observed. No
activation of the membrane is required. However, some proteins (<20 KD) may be lost
during post transfer washes.
Zeta-Probe positively charged nylon membrane allow binding of SDS protein complexes
in absence of methanol. These membranes are of choice when elution of high molecular
weight protein or protein having high negative charge is required. Small proteins bind
tightly. The capacity of Zeta-Probe nylon membrane (480 µg/cm2) is much higher as
compared to nitrocellulose membrane (80-100 µg/cm2). Blocking of membrane (Zeta-
Probe) is difficult and results in high background.