Western Blots

Immunoprecipitation analysis, as described in earlier portions of this chapter, has some fundamental limitations. It is seldom possible to know with certainty whether a radioactive band on a gel represents the polypeptide recognized by the antibody, or whether it is precipitated because it is physically attached to another (possibly nonradioactive) molecule which bears the antigenic determinant, A second problem with immunoprecipitation is that it may be difficult to radiolabel the antigen to sufficiently high specific activity.

These problems can sometimes be overcome by probing the proteins with antibodies after electrophoretic separation (Burnette, 1981). The procedure is often known as Western blotting (reviewed by Gershoni and Palade, 1983). The heterogeneous mixture of nonradioactive proteins is separated by electrophoresis in a polyacrylamide gel, and the proteins eluted electrophoret-ically by a transverse electric field onto a membrane which binds protein tightly. (Nitrocellulose is the most popular membrane.) The membrane is then probed with a radioactive antibody, washed and autoradiographed.

10.10.1 Sample Preparation for Western Blots

Some thought must be given to the preparation of the sample for Western blotting. If whole cells are lysed in SDS sample buffer, the resulting DNA

release may cause the solution to be very viscous and difficult to handle. Viscosity can be reduced to a manageable level by rapidly passing the lysate though a 21-gauge needle several times. The sensitivity of the method can often be greatly improved by preliminary enrichment procedures, such as immunoprecipitation or fractionation in Triton X-114. Dilute protein solutions may be concentrated by precipitation with organic solvents, which will also serve to remove nonionic detergents that will interfere with the running of SDS gels (see Section 10.7.3).

10.10.2 Enrichment of Antigen by Immunoprecipitation

It is possible to combine Western blotting with immunoprecipitation. The antigen is immunoprecipitated as above (see Section 10.8), and processed as usual for SDS-PAGE. The advantages of preliminary immunoprecipitation are greatly increased sensitivity and improved signal:noise ratio (Wiser and Schweiger, 1986). The disadvantage is that the gel will contain the immunopre-cipitating IgG, which will give a signal if anti-immunoglobulin reagents are used. This could be a major problem if the antigen of interest happened to co-migrate with IgG, but the problem can usually be overcome by judicious choice of reducing or nonreducing conditions to move the IgG to a part of the gel where it does not interfere with the desired signal.

10.10.3 Preliminary Enrichment for Membrane Antigens by Fractionation in Triton X-114

If the antigen is a membrane protein, fractionation of the antigen mixture in Triton X-114 provides an enrichment of about 20-fold (see Bordier, 1981, 1988; Brusca and Radolf, 1994). This simple procedure selectively partitions membrane proteins into a detergent-rich phase. It is simple, economical and effective. Details are given in Section 10.7.4. The presence of large amounts of Triton X-l 14 in the sample will cause SDS gels to run badly, and it is strongly recommended that the protein be precipitated with nine volumes of ice-cold acetone followed by centrifugation (5 min in Eppendorf centrifuge at 13 000 g) prior to resuspending in SDS sample buffer (see section 10.7.3).

10.10.4 Combination of Biotin Labelling, Western Blotting, and Enhanced Chemiluminescence

The enzymic generation of light has been used as an analytical tool for many years (reviewed by Whitehead et al, 1979). In 1983, it was discovered that the addition of certain substances, including phenylphenol, iodophenol or other aromatic compounds increase the sensitivity of luminescent assays by more than 1000-fold. The substrates are not particularly expensive, and the reaction is simple and convenient.

The advent of enhanced chemiluminescence (ECL) has had a major impact on the analysis of antigens, because it has made it possible to avoid the use of radioactive isotopes and at the same time to get results with shorter exposures. Instead of exposing autoradiographs for several days, exposure times can often be measured in minutes or seconds. The power of ECL is often exploited in conjunction with Western blots (see section 10.10.9).

The antigen mixture to be analysed is biotinylated (see Section 10.3), and then the desired antigen is isolated by immunoprecipitation. The precipitate is then dissociated and analysed by SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane, which is probed with peroxidase-strepta-vidin, followed by detection with ECL. Exposure times in the order of seconds to minutes are often possible. This approach is likely to become very popular (see Terashima et al., 1994, and Kim et al., 1994, for some elegant examples).

10.10.5 Western Blotting: Practical procedure

After electrophoresis, the gel is removed from the apparatus and placed in a dish containing 20% methanol in 20 raM Tris base, 150 mM glycine, pH 8. The methanol prevents the gel from shrinking or expanding. A sandwich is then built up, consisting of a perforated Plexiglass support, a porous polyethylene sheet (Bel-Art) or Scotch-Brite scouring pads, three sheets of heavy filter paper (Whatman 3M), the gel, the membrane, more filter paper, a porous pad and a second perforated support sheet (Fig. 10.10). All components should be wetted in buffer, and special care must be taken to avoid trapping air bubbles between the gel and the nitrocellulose membrane. The membrane must not be touched with ungloved fingers at any stage.

The whole assembly is held together with rubber bands, and placed in a tank containing buffer and platinum electrodes. Suitable tanks are available commercially, or can be improvized using plastic lunch boxes.

The current must be applied with the nitrocellulose membrane on the anode side of the gel. The field strength may be determined empirically, but typical values are 6-8 V/cm for 16-24 h (Burnette, 1981). The popular 'minigel' systems often require only 1 h for transfer.

Excessive current may result in overheating and distorted bands. If possible, transfer should take place in a cold room, with stirring using a magnetic 'flea' to prevent the build-up of pH gradients.

Some variability in the efficiency of transfer has been noted. In general, large proteins (Mr > 100000) may require higher field strengths and longer transfer times. Erickson et al. (1982) have shown that the addition of 0.1% SDS to the transfer buffer improves the transfer of high molecular weight o

Plexigla (perspex) shi o

Plexigla (perspex) shi

Perforated support Gel Nitrocellulose sheet

Fig. 10.10. Western blotting. The polyacrylamide gel containing the sample is overlaid with a sheet of nitrocellulose, and sandwiched between filter papers, kitchen scouring pads and perforated Perspex (Plexiglass) supports. The whole sandwich is held together by rubber bands, and submerged in a buffer consisting of 20 mM Tris base, 150 mM glycine and 20% methanol in water. The methanol prevents the gel from shrinking or expanding. An electric field is applied such that the nitrocellulose is on the anode side of the gel. Proteins in the gel are electrophor-etically transferred to the nitrocellulose, to which they bind.

Perforated support Gel Nitrocellulose sheet

Platinum wire

Fig. 10.10. Western blotting. The polyacrylamide gel containing the sample is overlaid with a sheet of nitrocellulose, and sandwiched between filter papers, kitchen scouring pads and perforated Perspex (Plexiglass) supports. The whole sandwich is held together by rubber bands, and submerged in a buffer consisting of 20 mM Tris base, 150 mM glycine and 20% methanol in water. The methanol prevents the gel from shrinking or expanding. An electric field is applied such that the nitrocellulose is on the anode side of the gel. Proteins in the gel are electrophor-etically transferred to the nitrocellulose, to which they bind.

proteins. An additional source of variability is the brand and batch of the nitrocellulose. Some brands are treated with a nonionic detergent, which inhibits protein binding. Washing the membrane in distilled water or transfer buffer prior to use often improves the transfer.

10.10.6 Blocking the Western Membrane

After the transfer of proteins from the SDS gel, the membrane must be blocked so that further nonspecific binding of protein is prevented. Nonfat powdered milk (Bovine Lacto Transfer Technique Optimizer or BLOTTO) is a very effective substitute for BSA, which was previously the reagent of choice (Johnson et al, 1984). BLOTTO consists of 5% (w/v) nonfat powdered milk in PBS plus 0.01% Antifoam A (Sigma) and 0.001% merthiolate as preservative. If the BLOTTO is used on the day that it is made, the merthiolate is unnecessary. The Antifoam is also optional.

Results using BLOTTO are generally much better than those using other blockers, and the cost is much less than BSA. BLOTTO is now the reagent of choice for blocking. BLOTTO may also substitute for BSA in other immunological procedures such as radioimmunoassays.

Many other blocking agents have been used, including high concentrations of BSA or casein. Batteiger et al. (1982) have shown that the detergent Tween 20 may be used as a blocker, but the use of nonionic detergents is risky as it may cause serious losses of protein from the membrane. Polyvinyl alcohol is said to be an effective blocker, and to act virtually instantaneously (Miranda et al., 1993).

10.10.7 Probing the Membrane

After blocking, the membrane is placed in a dish containing the antibodies (typical concentration 1-50 pg/ml) in BLOTTO. After 30-60 min, the membrane is removed, washed in BLOTTO, and placed in the same buffer containing an appropriate dilution of peroxidase-conjugated anti-immunoglobulin or protein A, or 105 c.p.m./ml of 125I-labelled staphylococcal protein A or affinity-purified anti-immunoglobulin. After a further 30-60 min, the membrane is removed, washed extensively, and processed for autoradiography.

10.10.8 Readout for Western Gels

Readout by ECL (see Section 10.10.9) is now the preferred method, as it is extremely sensitive and often requires only a few seconds to obtain adequate sensitivity. If the background staining of the membrane is excessive owing to the extreme sensitivity of ECL, dilution of the ECL substrate may be helpful (see Pampori et al., 1995).

Alternatively, 125I-labelled anti-immunoglobulin or protein A may be detected by autoradiography using intensifying screens (Fig. 10.7). The use of colour readout by ELISA is no longer recommended as it lacks sensitivity and often fades upon storage.

It has recently been reported that the sensitivity of detection of unreduced biotinylated proteins in Western blots may be greatly increased by soaking the gel in (3-mercaptoethanol prior to transfer (Weston et al., 1995). This is probably due to improved access of avidin to the biotin rather than improved efficiency of transfer.

The potential sensitivity of Western blotting is extremely high. Burnette (1981) has shown that is easily capable of detecting murine leukemia virus proteins from as few as 1000 cells. When used with ECL, even greater sensitivity would be possible.

10.10.9 Enhanced Chemiluminescence—Practical Procedure

Enhanced chemiluminescence systems are available using horseradish peroxidase- or alkaline phosphatase-conjugated antibodies or streptavidin.

Appropriate substrate mixtures are available commercially. Autoradiography is performed using blue-sensitive film without intensifying screens. Kits are available from Amersham, Dupont, Boehringer, Bio-Rad and other suppliers.

The following information is for the Amersham system. The membrane is probed with peroxidase-conjugated antibody or peroxidase-conjugated strep-tavidin, washed in PBS/BLOTTO, and washed twice more in PBS without BLOTTO. From this point on, one should work quickly, preferably in a dark room close to the X-ray developer. The membrane is placed in a plastic bag or Petri dish containing a freshly prepared mixture of each of the two ECL solutions (typically 125 fil/cm2 of membrane), and incubated for precisely 1 min at room temperature. The reagents are then drained off. The membrane, while still damp but not wet, is placed between two plastic sheets and exposed to X-ray film with the protein-containing side facing the film.

If horseradish peroxidase is used, the light emission peaks at about 20 min, and slowly declines over a period of hours. The duration of light emission is somewhat longer if alkaline phosphatase system is used, but the relative sensitivity of the two systems has not been rigorously compared. As the turnover number for horseradish peroxidase is greater than alkaline phosphatase, the sensitivity for the former may be greater.

It will usually be necessary to take several exposures. The first exposure could be typically about 1 min, and subsequent exposures should be guided by results.

10.10.10 Use of Remazol Prestained Molecular Weight Standards with Western Blots

It will often be desirable to identify the molecular weight standards on Western blots, but it is not possible to use Coomassie blue to stain nitrocellulose blots, because the membrane binds the dye. Bound proteins can be stained with Ponceau red, but this stain is not very sensitive. A much better approach is to use Remazol blue stained standards, in which the dye is cova-lently attached to the proteins (Griffith, 1972). In this way, the prestained standards can be seen while the gel is running. The prestained standards can also act as a very useful guide for alignment of the gels and cutting up the membrane. The mobility of the Remazol stained standards is not quite the same as for unconjugated standards but the difference is minor. The sensitivity of Remazol is not as high as Coomassie blue, and slightly more protein will have to be added to the gel.

Remazol brilliant blue R (Sigma cat. no. R-8001, 25 or 100 g bottles) is a textile dye, and is the potassium salt of a sulfatoethyl sulfonic acid. It reacts with primary and secondary amines, alcoholic OH groups and SH groups. Binding is covalent. Coupling is inhibited by amines (e.g. Tris), thiols and hydroxyl-containing components (e.g. glycerol). These must be absent from the reaction buffer.

The dye solution is made up by dissolving 10 mg Remazol brilliant blue in 1.0 ml 10% SDS in water. The coupling is carried out in 0.2 M carbonate/bicarbonate buffer, pH 9.5 (0.86 g Na2C03 plus 1.72 g NaHCOj to final volume of 100 ml distilled water; the pH should be about 9.3-9.5 without titration). Both the dye solution and the buffer can be stored frozen for subsequent use.

For low molecular weight standards, dilute two vials of Pharmacia low molecular weight standards (cat. no. 17-0446-01) into a total of 180 ¡j.1 pH 9.5 coupling buffer. For high molecular weight standards, take 100 of the BioRad HMW stock (cat no. 161-0303; supplied in 50% glycerol) and dilute to a final volume of 200 jxl with water. Remove the glycerol from the Bio-Rad standards by overnight dialysis against two changes of 1 1 of coupling buffer. Failure to dialyse out all the glycerol will result in failed coupling.

Practical procedure

(1) Add 50 jxl Remazol mixture to the standard solution prepared as above.

(2) Heat at 70°C for 30-60 min (not critical). Dilute to 1 ml with reducing SDS sample buffer. Nonreduced standards are slightly less accurate and give more diffuse bands.

10.10.11 Western blots with Nonspecific Polyclonal Antisera

If the relevant antigen is available in pure form, or if it has detectable enzymic activity, it is possible to adapt the Western blot so that the antigen may be identified even when the antiserum contains numerous irrelevant antibodies (Muilerman et al., 1982; van der Meer et al., 1983). The gel is run and blotted as usual, and probed with the nonspecific antiserum. Instead of antiimmunoglobulin or staphylococcal protein A, the membrane is probed with 125I-labelled pure antigen or the enzyme-containing mixture. After washing, the antigen is detected by autoradiography or by enzyme assay. The fact that antibodies are symmetrical (with two identical binding sites) means that any antibodies that have only one site bound to the nitrocellulose have one free site which allows the labelled antigen or enzyme to bind. Only the subpopulation of specific antibodies are thus detected.

10.10.12 Use of Western Blots with Monoclonal Antibodies

It is usually considered that proteins which have been heated to 100°C in SDS plus reducing agents are totally denatured. This view may not be strictly correct. Immunoglobulin y-chains isolated from SDS gels and subjected to limited proteolysis in SDS-containing buffers were cleaved almost exclusively at regions between domains (Goding, 1982). The existence of many additional potential cleavage sites was revealed by the substantially larger number of breakdown products when digestion was performed in 4 M urea.

The conformations that proteins adopt after they have bound to nitrocellulose are not well understood, nor is the precise mechanism of binding to the membrane. It would seem likely that proteins bound to the membrane would be electrophoretically 'stripped' of bound SDS, which would continue to the anode. Accordingly, bound proteins might thus regain more of their native configuration.

It has been known for many years that the reactivity of immune serum made against native proteins is usually much weaker when tested on denatured proteins (see Chapter 4 for discussion). However, antisera against the native protein will almost always contain at least some clonal products that recognize the denatured protein, and vice versa. It is this fact that allows the Western technique to function.

In marked contrast to polyclonal antibodies, monoclonal antibodies against the native protein may or may not recognize the denatured product, in an all-or-none fashion. It may be expected that many clones (perhaps the majority) will fail to recognize the denatured antigen. It would be expected that the Western technique would work much more reliably with polyclonal antibodies, and experience is consistent with this view. None the less, Western blots sometimes work with monoclonal antibodies, and are well worth trying.

Goldstein et al. (1982) produced a monoclonal antibody against cytomegalovirus, which detected an Mr 80000 protein in Western blots. Interestingly, the antibody did not precipitate any protein in standard immunoprecipitation system, perhaps because it only recognized the denatured form of the antigen. Barbour et al. (1982) were able to use the Western technique with a considerable proportion of monoclonal antibodies against variable proteins of Borrelia hermsii. Parham et al. (1982) found three out of 15 monoclonal anti-HLA antibodies worked in Western blots. O'Connor and Ashman (1982) found four out of five monoclonal anti-Salmonella antibodies detected protein in Western blots.

The chances of a monoclonal antibody working on Western blots are improved if the sample is analysed without reduction, presumably because intact disulfide bonds prevent complete unfolding and may help guide refolding. It may also help to avoid boiling the sample.

10.10.13 Recovery of Antibody, Antigen and Re-use of Western Blots

Antibody may be eluted off the nitrocellulose-immobilized protein by treatment with pH 2.2-2.5 glycine-HCl, preferably in the presence of 0.1-1.0%

BSA (Legochi and Verma, 1981; Erickson et al., 1982). The nitrocellulose-immobilized protein remains firmly attached to the nitrocellulose. Indeed, it cannot be removed by SDS or deoxycholate, although it is efficiently eluted by Triton X-100, Nonidet P-40 or octyl glycoside (Lin and Kasamatsu, 1983). The general rule appears to be that nonionic detergents are good blockers and eluters, while anionic detergents neither block nor elute, perhaps because of repulsion from the negatively charged paper. The bound protein can also sometimes be recovered by dissolving the nitrocellulose in dimethyl sulfoxide, and may be used as antigen for immunization (Knudsen, 1985).

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