Western blotting

4.11 Western blotting

A sheet of nitrocellulose is placed against the surface of an SDS-PAGE protein fractionation gel (see Appendix B.2.1) and a current applied across the gel (at right angles to its face), thus caus- ing the proteins to move out of the gel and onto the nitrocellulose where they bind firmly by covalent forces. Two variants of the basic apparatus are available to achieve the electrophoretic transfer, the so-called wet and semidry blotters: wet blotters: the gel and transfer membrane are immersed in large volumes of buffer; semi-dry blotters: use filter paper pads moistened with buffer.

4.11.1 Immuno- or Western blotting of SDS-PAGE

MATERIALS AND EQUIPMENT Wet blotting apparatus (see Appendix C)

Polyacrylamide running buffer, 0.1 M tris(hydroxymethyl)-aminomethane (Tris)–glycine, pH 8.3, with 500 ml of methanol added to 2 l of blotting buffer (see Appendix A) Nitrocellulose paper Whatman no. 1 filter paper Scotch-brite pads

METHOD

1 Equilibrate the SDS-PAGE gel (containing the fractionated protein) in blotting buffer for 30–60 min.

2 Assemble the blotting sandwich within the blotting cassette taking, in order, Scotch-brite pad, nitrocellulose sheet, polyacrylamide gel and Scotch-brite pad (anode → cathode) (Fig. 4.7). Take care to avoid any air bubbles between the gel and the nitrocellulose.

3 Insert the cassette into the blotting buffer and connect the power supply. The cathode should be on the gel side.

4 Blot for about 3 h at 60 V or overnight at 35 V.

4.11WESTERN BLOTTING

Wet blotting assembly

– Stainless steel

cathode

+ Palladium

Support pad

anode

(household scouring pad)

SDS-PAGE gel

Nitrocellulose

Support pad

sheet

Semi-dry blotting assembly

CATHODE

Six filter papers soaked in cathode buffer

Repeat with multiples of the sandwich for two or more gels

Fig. 4.7 Assembly sequence for

Dialysis

sandwich for wet and semi-dry

membrane

blotting. The precise details of the components will vary with the apparatus used; however, the order

GEL

Nitrocellulose

of assembly aanode, nitrocellulose

Blotting sheet, SDS-PAGE gel, cathode ais sandwich

sheet

vital. If it is reversed by mistake, the for one gel

Filter papers

soaked in:

carefully separated proteins will be

Anode buffer 2

electrophoretically transferred to 5 l of tank buffer. The semi-dry blotter not

Anode buffer 1

only avoids the use of large volumes of buffer but also can be run as a sandwich

ANODE

of several gel–nitrocellulose sheets interspersed with low molecular weight cut-off dialysis membranes. The dialysis

membrane permits the passage of ions but not of proteins.

TECHNICAL NOTES • Methanol is used in the blotting buffer to prevent the gel swelling. • Take great care to ensure that the blotting cassette is assembled in the correct order. Proteins

transferred into the filter pads rather than the nitrocellulose will be lost. • It is good practice to stain the gel for proteins after blotting over, to ensure that the transfer was complete. Some proteins, e.g. the relatively insoluble cytoskeletal components, tend to transfer inefficiently and non-quantitatively.

• Blots may be dried, sealed in a plastic bag and stored at –20°C for up to 6 months.

C H A P T E R 4: Antibodies as probes

4.11.2 Staining Western blots

Blots may be stained with the usual protein stains. If it is intended to visualize the polypeptide bands by deposition of enzymically generated colours, 0.02% Ponceau S in 3% trichloroacetic acid can be used as a temporary stain, but may cause some protein denaturation. The blots are differentiated in running tap water. This stain is completely reversible and removed by washing in tap water.

4.11.3 Blocking protein-binding sites

Before visualization of protein bands with labelled antibodies it is necessary to block the remain- ing protein-binding sites on the nitrocellulose. Bovine serum albumin, haemoglobin or skimmed milk powder are frequently used for blocking, sometimes with the addition of 0.05% Tween 20. Tween 20 alone may be used and has the advantage that, after visualization with specific antibod- ies, the whole blot can be stained with protein stains. Moreover, the added advantage is that it reveals the markers for molecular weight determination, allowing the visualization of protein bands which have failed to bind antibody. A word of caution: Tween 20 alone is not an efficient blocking agent, so when concentrated protein solutions are used, such as serum or ascitic fluid, the background can be unacceptably high.

MATERIALS AND EQUIPMENT Phosphate-buffered saline (PBS), containing 0.05% Tween 20 and 1% w/v bovine serum albumin

(BSA) Plastic box

METHOD

Place the nitrocellulose blot in the PBS–Tween–BSA and gently rotate on an orbital shaker for about 2 h at room temperature, or overnight in the cold.

4.11.4 Immunostaining nitrocellulose blots

Although proteins separated by SDS-PAGE and blotted onto nitrocellulose have been subjected to harsh denaturing conditions (detergent treatment, boiling, reducing agents and distortion due to adsorption to the highly charged surface of the nitrocellulose membrane) a large proportion of antigenic determinants remain intact and can be revealed by the addition of labelled antibodies. In the past, radiolabelled antibodies or antibodies detected by radiolabelled protein A have been extensively used. Although the technique offers high sensitivity, preparation for autoradiography can be complex and time consuming, and carries the risk of chronic exposure to low levels of radioactivity. The techniques for the use of enzyme-labelled antibodies have been improved greatly and a range of substrates developed which are not carcinogenic and can be used to deposit

a variety of coloured, insoluble products on top of the protein band.

Biotinylated antibodies followed by streptavidin–peroxidase give well visualized bands with 4-chloro-1-naphthanol as substrate. Carbohydrate determinants maintain their immuno- genic properties after SDS-PAGE fractionation and can be detected with biotinylated lectins or antibodies.

4.11WESTERN BLOTTING

MATERIALS Nitrocellulose blot with immunoglobulin bands Biotinylated antibody, e.g. biotinylated goat anti-human IgG streptavidin–peroxidase Phosphate-buffered saline (PBS) containing 0.05% v/v Tween 20 with and without 1% w/v bovine

serum albumin (BSA) (PBS–Tween–BSA) 4-chloro-1-naphthanol

0.05 M tris(hydroxymethyl)-aminomethane (Tris)–HCl buffer, pH 7.6 Hydrogen peroxide

METHOD

1 Prepare a dilution of biotinylated antibody in PBS–Tween–BSA; as a guide, high-titred antibodies can be used at a dilution of 1 : 1000. Prepare a sufficient volume of diluted antibody to cover the blot.

2 Immerse the blot in the antibody solution and agitate gently for 2 h on an orbital shaker.

3 Wash the blot thoroughly by immersing in excess PBS–Tween (0.05%); no BSA. Mix gently for 10 min on the orbital shaker.

4 Repeat the wash step three more times.

5 Add sufficient streptavidin–peroxidase conjugate (diluted 1 : 1000 in PBS–Tween–BSA) to cover the blot. Mix gently for 1 h on the orbital shaker.

6 Wash the blot by immersing in four changes of PBS–Tween as before.

7 During the final wash prepare the substrate solution by dissolving 6 mg 4-chloro-1- naphthanol in 20 ml methanol and adding it to 100 ml Tris–HCl buffer, pH 7.6, plus 12 µl hydrogen peroxide (30 volumes’ strength).

8 Immerse the washed blot in the substrate solution. Mix gently on the orbital shaker. Bands should develop sufficient colour within a few minutes.

9 Remove the blot and wash with distilled water.

10 Dry the blot and analyse immediately. Although the coloured bands fade with time, the rate of colour loss can be retarded if the blots are kept in the dark.

TECHNICAL NOTES • If the antibody is expensive, or the supply limited, a smaller volume of diluted antibody may be

applied to the nitrocellulose sheet by means of saturated filter paper. It is essential to achieve good contact between the paper and the nitrocellulose. This may be achieved by sealing the nitrocellulose–filter paper sandwich into a plastic bag or by clamping it between two glass slides.

• Some of the substrates still in use are known carcinogens, e.g. bisdiazotized benzidine. These should only be handled in a fume cupboard. • The colour reaction can be quantified by scanning densitometry and a permanent record made by photography, e.g. the Biogene Documentation System.

4.11.5 Western blotting with radiolabelled probes

1 Proceed as method above but at step 5 add 125 I-streptavidin instead of the enzyme-labelled

material. Use about 1 × 10 6 c.p.m. per track.

2 After washing, dry the blot for 10–20 min between filter papers.

C H A P T E R 4: Antibodies as probes

3 Wrap in cling film and insert into an X-ray cassette with autoradiographic film.

4 Leave the autoradiograph to expose at –70°C for between 12 h and 1 week depending upon the amount of radioactivity bound. Develop the film according to the manufacturer’s instructions.

TECHNICAL NOTES • The sensitivity of the detection system may be increased using:

(a) Preflashed film. The duration of the flash and its intensity (varied by changing the distance between film and electronic flash-gun) is critical and should be determined empirically. Set the shortest duration on the flash-gun and position the gun at varying distances (increasing in steps of 1 m) from film test strips. Develop the film and use the conditions that just give barely visible fogging. Exposure of preflashed film at −70°C gives maximal stability of both the nascent image and the sensitized preflashed areas.

(b) Intensifying screens. Screens should be placed on either side of the blot–film sandwich. The screens are impregnated with heavy metal ions coupled to a scintillation system. The efficiency of capture of the emitted radiations is therefore increased and more of them are recorded on the X-ray film because of the photon emissions from the screens. Screens are important for the efficient capture of 131 I.

• The system described above may also be adapted for analysis using 125 I-labelled antibodies or unlabelled antibodies detected by 125 I-labelled protein A.

4.11.6 Chemiluminescence as a non-radioactive method for detecting antigens by Western blot analysis

This non-radioactive method is available for detecting antigens by Western blot analysis. This utilizes a chemiluminescent reagent which reacts with enzyme-labelled antibodies, e.g. horse- radish peroxidase. Here is an outline of the basic technique. • Horseradish peroxidase oxidizes a peracid salt which then leads to a raised oxidation state of

the haem group within the horseradish peroxidase itself. • As this raised state decays, luminol radicals are formed, which on decaying emit light. • In an enhanced assay, enhancer phenolic molecules are placed between the haem group and

the luminol which results in increased (1000-fold) and prolonged emission of light with a half-life of around 60 min. The above technique has been further developed by Lumigen Inc. with the enzymatic genera-

tion of an acridinium ester which produces an intense light emission athis is the more sensitive alternative ECL Plus (Code RPN 213, available from AP Biotech) where the light emission is of longer duration than the original ECL system. Both the ECL and ECL Plus system can be used on either nitrocellulose (Hybond C) or polyvinylidene difluoride (PVDF) (Hybond P) blotting membranes.

MATERIALS Non-fat dried milk powder Phosphate-buffered saline (PBS) containing 0.1% (v/v) Tween 20 Tris(hydroxymethyl)-aminomethane (Tris)-buffered saline, pH 7.6 PBS, pH 7.5 Nitrocellulose membrane Primary antibody (raised against antigen of interest)

4.11WESTERN BLOTTING

Horseradish peroxidase (HRP)-conjugated second antibody (to react with the primary antibody) Streptavidin biotinylated HRP conjugate ECL reagents 1 and 2 X-ray film

METHOD

1 Prepare and electrophorese antigens on the SDS-PAGE gel, then blot by the usual protocol.

2 Block the nitrocellulose membrane with PBS, pH 7.5, containing 0.1% (v/v) Tween 20 and 5% (w/v) non-fat dried milk powder.

3 Leave the membrane in the blocking solution overnight at 4°C.

4 Wash the membrane with 3 × 2-min washes of PBS–Tween 20.

5 Dilute the primary antibody to the appropriate dilution in PBS–Tween 20.

6 Incubate the washed membrane with the first antibody solution at 20°C with continuous shaking for 60 min.

7 Wash the membrane with 2 × 2-min washes of PBS–Tween 20.

8 Dilute the second antibody (biotinylated or HRP-labelled) in PBS–Tween 20.

9 Incubate the membrane with the HRP-labelled second antibody solution at 20°C with continuous shaking for 60 min.

10 Serially wash the membrane in PBS–Tween 20 at 20°C for:

2 × 1 min

1 × 15 min

3 × 5 min. Note: If using HRP-labelled antibody, go to step 13.

11 Make the working dilution of the biotinylated streptavidin–HRP complex in PBS–Tween 20,

then incubate with the membrane at 20°C with continuous shaking for 60 min.

12 Serially wash the membrane in PBS–Tween 20 at 20°C for:

13 Substrate preparation. The peracid salt is kept separate from the luminol/enhancer mixture and mixed just prior to use. Mix equal volumes of reagent 1 and 2 (ECL working solution).

14 Lay blot flat on cling film (Saran wrap) with the protein side uppermost.

15 Pipette ECL working solution on to the blot and leave for 1 min.

16 Drain and wrap in cling film.

17 In a dark room, place the wrapped blot against the X-ray film and expose for 15 s (longer or shorter times may be necessary).

18 Develop using standard X-ray developing chemicals. TECHNICAL NOTES

• The pH will need to be adjusted to pH 7.5 with concentrated sodium hydroxide when the milk powder has been added. • BSA may not be used as a blocking agent with the ECL Plus detection reagents. • The incubation with the dried milk powder is for blocking any non-specific binding sites. • Obviously the dilution of the antibody (first and second) will vary, depending on the antigen–

antibody system being used. A ‘typical’ polyclonal antibody may work at around 1 : 10 000.

C H A P T E R 4: Antibodies as probes

Therefore it is advisable to perform the first assay with a reasonably low dilution of antibody and then make further dilutions up or down, depending on the results obtained. If using the ECL HRP-conjugated antibodies, the recommended dilution range is 1 : 3000–1 : 12 000.

• Following ECL detection, it is possible to reprobe the membrane several times.