biochemical techniques biochemistry 21 western blotting

Biochemistry Biochemical Techniques

21 Western Blotting


21 Western Blotting

Description of Module

Subject Name Biochemistry

Paper Name 12 Biochemical Techniques

Module Name/Title 21 Western Blotting


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


21 Western Blotting

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


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


21 Western Blotting

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


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


21 Western Blotting

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


21 Western Blotting

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-


21 Western Blotting

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


21 Western Blotting

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


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).


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.

biochemical techniques biochemistry 21 western blotting

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