.lournal of Virological Methods, 30 (1990) 25-40 25

Elsevier

VIRMET

01064

Protein blotting: ten years on David R. Harper, Kit Ming-Liu* and Hillar 0. Kangro Vim&y

Department, St. EurrhuIo~ew’s (Accepted

5 June

Hospital, London. ff.K. 1990)

Summary Protein blotting was originally described in 1979 as an outgrowth of nucleic acid techniques, and received its commonly used designation of ‘Western’ blotting in 1981. The use of the technique to render electrophoresed proteins accessible for further analysis has found many roles, the most prominent being subsequent reaction with antibodies or antisera, which has many clinical and research applications. Since the initial development of the system there have been many changes to the techniques involved, but the basic principles remain unaltered. This review discusses these changes, and also provides a summary of current techniques. Blotting; Western blotting; Immunoreactivity

Since its initial development as an outgrowth of DNA (Southern) and RNA (Northern) blotting (Alwine et al., 1977; Southern, 1973, protein blotting has been evolving constantly. As with DNA blotting, the basic principle is the transfer of electrophoresed material from the gel matrix onto a membrane which binds the eluted macromolecules, allowing subsequent reaction with specific probes. For DNA, the elution is from an agarose gel by flow of buffer through the gel onto a membrane (usually nitrocellulose or nylon), where the DNA is retained. The probes are nucleic acids with sequences complementary to the bound molecules. While there are many similarities to the methods used in protein blotting, the two procedures are by no means identical. Protein separations usually use polyacrylamide gels, which have a much Correspondence West Smithfield, *Current

to: D. Harper, Department of Virology, London ECIA 7BE, U.K.

address:

0168~8510/90/$03.500

Virology

Department,

St. Thomas

1990 Elsevier Science Publishers

Medical Hospital,

College London.

B.V. (Biomedical

of St, Bartholomew’s U.K. Division)

Hospital,

26

greater sieving effect than the agarose gels normally used for DNA. As a result, early protein transfers using passive buffer flow elution similar to that used for DNA (Renart et al., 1979) gave very poor elution, unless this difference was compensated for. Various attempts were made to improve elution, including breakup of the gel matrix after electrophoresis by the use of variant crosslinkers (Renart et al., 1979) and proteolysis of the electrophoresed proteins (Gibson, 1981), but it was with electrophoretic mobilisation of the proteins from the gel matrix that protein blotting became a practical technique. While Bittner et al. (1980) used a system of electrophoretic transfer with a sodium phosphate buffer, Towbin et al. (1979) described transfer to a nitrocellulose membrane using a Tris/glycine/methanol buffer system in a tank apparatus, followed by reaction with antisera and protein A, a staphylococcal membrane protein which selectively binds immunoglobulins. The procedure of Towbin et al. (1979) was widely adopted, and is basically similar to current methods. Burnette (1981) coined the term ‘Western blotting’ to describe a slightly modified form of this technique, thereby retaining the ‘geographic’ naming tradition initiated by Southern (1975). This term is now used to describe almost all forms of protein blotting, even those which differ substantially from that of Bumette, although the terms immunoblotting or electroblotting are also used. This review discusses the evolution and applications of the technique, and of the methods and equipment involved. The names of suppliers given in the text are examples based on the experience of the authors, and are not intended to represent endorsements of the listed products or definitive listings of suppliers.

Blotting

systems

The original form of the blotting platinum wire electrodes, into which (Fig. la), and such systems may still LKB). However, variation in intensity

apparatus was a buffer-filled tank with the gel-membrane sandwich was inserted be obtained (Bio-Rad; Hoefer; Pharmacia of the electrical field due to the shape of

Filter paper (2 sheets) AbSOrbent pad

Buffer tank

-filled

Weight

Gel M,embrane Filter paper (9 sheets)

Fig. 1.

27

TABLE 1 Comparison of typical tank and semi-dry blotting systems Method

Tank blotting

Semi-dry blotting

Electrodes Transfer time Buffer required Maximum capacity External cooling required

Platinum wire 16 h 2-3 1 2 gels Yes

Conductive l-3 h

plate

I I 6 gels No

the electrodes has been reported to produce significant localised variations in the efficiency of transfer (Gershoni, 1985; Gershoni ‘et al., 1985b). This problem is avoided by the use of solid electrodes, either in a buffer tank or in a ‘semi-dry’ blotting apparatus. This latter method does not use buffer in a tank, but rather a stack of buffer-saturated filter paper placed between two solid electrodes (Fig. lb). With a tank blotting system, transfer is typically of a maximum of two gels (although up to nine may be transferred in some systems), and normally requires at least 16 h. Semi-dry blotting allows the simultaneous transfer of up to six gels and uses far less buffer. In addition, transfer time is greatly reduced with a transfer being completed in one to three hours, although this may be extended if required. These differences are summarised in Table 1. Semi-dry blotting may well be the most significant technical development in protein blotting since the original application of electrotransfer. The original semi-dry blotting procedure (KhyseAndersen, 1984) used graphite plate electrodes and an unusual isotachophoretic transfer system. This method has been largely superseded by more traditional electrophoretic transfer, and there have been significant developments in the construction of electrodes. Oxidation of the anode prevents the use of reactive metals for this purpose (Svoboda et al., 1985), and a wide range of materials have been used. Svoboda et al. (1985) used stannous oxide coated surface conductive glass as the anode, with a stainless steel cathode. More recently, various proprietary blotting cells have appeared, using graphite (Dako; Genetic Research Instrumentation; Pharmacia LKB; Sartorius), glass/steel (Bio-Lyons), platinum/steel (Bio-Rad; Hoefer), or conductive plastic electrodes (Millipore), and no standard construction has yet been established. Some workers have elected to use differing anodic and cathodic buffers to enhance recovery (Svoboda et al., 1985), but this usually limits the transfer to one gel at a time, which reduces the usefulness of the method. Electroblottirig has also been used to transfer proteins after isoelectric focussing (Matthei et al., 1986) and two-dimensional electrophoresis (Dunn et al., 1987; Jackson and Thompson, 1984) extending the uses of the technique. In all blotting systems, great care must be taken to exclude air bubbles between the gel and the membrane,. since these will interfere with blotting, resulting in localisdd areas of poor transfer.. Air bubbles should be gently expelled from the side of the’gel-membrane sandwich using a wettened roller (often a pipette) in order to avoid this problem.

28

Buffers The basic formulation of blotting buffer has changed little. The presence of methanol in the transfer buffer increases the binding of proteins to the nitrocellulose, reduces gel swelling, and aids cooling during the transfer. However, it also contracts the pores of the gel, reducing elution of proteins (Gershoni and Palade, 1982; Nielsen et al., 1982; Szewczyk and Kozloff, 1985). The use of low levels of SDS in the range of 0.01 to 0.02% (w/v) can counteract this latter effect and has also been noted to increase transfer efficiency, particularly of high molecular weight proteins (Harper et al., 1988; Nielsen et al,, 1982; Perides et al., 1986), but may also adversely affect subsequent immunoreactivity (Birk and Koepsell, 1987). A transfer buffer consisting of 192 mM glycine, 25 mM Tris base, 20% (v/v) methanol,,0.02% SDS, pH 8.3, provides a good overall transfer efficiency in most systems. It does appear to be an unavoidable aspect of protein blotting that the transfer efficiency of proteins is inversely proportional to the molecular weight (Bumette, 1981; Lin and Kasamatsu, 1983). The specific properties of the protein being transferred may also affect its elution, for example with strongly basic proteins (Szewczyk and Kozloff, 1985), requiring alterations to the conditions of transfer.

Membranes The selection of a protein-binding membrane is a key step in the procedure. Some early work used diazobenzyloxymethyl paper (Bittner et al., 1980; Renart et al., 1979), as used in RNA blotting, but nitrocellulose was found to give improved resolution with a less complex procedure (Bumette, 1981). Nitrocellulose is widely available (Amersham; Bio-Rad; Millipore; Pharmacia LKB; Schleicher and Schuell), and is the most commonly used blotting membrane, giving reliable and reproducible results, but with certain disadvantages. Retention of low molecular weight proteins can be poor unless unusually small pore sizes are used (Lin and Kasamatsu, 1983), and the membrane is fragile, particularly when dry. Therefore, it is not surprising that (usually more expensive) alternatives have been developed. Activated (charge-modified) nylon membranes (Amersham; Bio-Rad; Pharmacia LKB) are more commonly used for DNA blotting, but have been used for proteins (Gershoni and Palade, 1982). Nylon membranes have a high binding capacity and are extremely strong, but require rigorous blocking (see below) and only a limited selection of protein stains may be used due to the positive charge present on the membrane (Gershoni and Palade, 1982). Nylon-supported nitrocellulose membranes are available (Amersham), which give some of the strength advantagesof nylon, while binding properties are conferred by the nitrocellulose component.. Another approach is the development of an entirely new material, and this approach has resulted in the polyvinylidene difluoride (PVDF) membrane (Millipore; Schleicher and Schuell). This has greater mechanical strength than nitrocellulose, but avoids the blocking and staining problems associated with

29

nylon (Pluskal et al., 1986). However, a possible question mark over the use of this membrane must be the ease with which bound proteins are eluted into mild detergent solutions (Szewczyk and Summers, 1988), and wetting of unblocked PVDF membrane requires the use of high methanol concentrations.

Shining It is often desirable to visualise the transferred protein to allow exact alignment and for calibration purposes. Staining of a section containing molecular weight markers allows reliable determination of the molecular weight of bands detected by immunoreaction; an interesting alternative approach is to use antisera against the markers in an immunoreaction (Carlone et al., 1986). However, staining of the whole gel provides a form of quality control, allowing areas of poor transfer to be located and avoided. There are two basic approaches to staining: The proteins may be stained before, or after, the transfer takes place. A number of stains are available, of varying sensitivity. Initially, the stains used for visualisation of proteins in gels were also used for nitrocellulose, typically Coomassie blue or amido black (Lin and Kasamatsu, 1983), although these stains cannot be used with nylon membranes, since the charged nature of the membrane results in very high levels of background staining (Gershoni and Palade, 1982; Pluskal et al., 1986). India ink staining has been reported to be more sensitive (Hancock and Tsang, 1983), and may also be used with nylon (Hughes et al., 1988), while high resolution gold stains are available commercially. Alternatively, stains specific for particular chemical groups within the bound proteins, such as sulphydryl groups (Bayer et al., 1987), may be used where the group in question is of particular interest. Amido black can be completely removed from nitrocellulosebound proteins (Harper et al., 1986), as can the red dye Ponceau S (Salinovich and Montelaro, 1986). This can be useful, as it allows staining to be used prior to the use of ch~mogenic detection systems. Staining prior to electroblotting has been reported primarily with Coomassie blue (Jackson and Thompson, 1984; Jackson et al., 1985; Perides et al., 1986), although the use of other stains has been reported (Tracy et al., 1987). One problem with this approach is the longer staining time required with proteins contained in the gel matrix. However, the major problem with both approaches is that staining frequently results in a reduction in immunoreactivity of the membrane-bound proteins. This may be due to the blocking of antibody binding by the protein-bound stain molecules, or to the denaturing effects of solvents during the staining process (Tracy et al., 1987). Various techniques have been suggested to minimise the effects of staining on immunoreactivity, and it appears that in particular applications the effects of staining can be negligible (Glenney, 1986; Harper et al., 1986; Tracy et al., 1987). No staining technique has yet been shown to be completely free of this problem, and it is a matter for debate whether the advantages of staining are sufficient to compensate for its effects. However, many of the techniques which are involved in protein blotting have similarly damaging effects on subsequent

30

immunoreactivity (see below), and while staining can reduce immunoreactivity, this must be viewed in the context of the pre-existing denatured. state of the proteins.

Blocking After transfer (and staining) is complete, unused macromolecular binding sites must be blocked to prevent non-specific adsorption of probe molecules. The selection of a blocking solution from the wide range available, together with the temperature and duration of the blocking incubation, may affect the level of background staining (Hauri and Bucher, 1986; Thean and Toh, 1989), and it is impo~~t that this step is optimised for the system in use, The majority of blocking solutions. are protein-based (Hauri and Bucher, 1986; Thean and Toh, 1989), although use of a solution of Tween-20 non-ionic detergent has been reported (Baettiger et al., 1982). Buffered solutions of skimmed milk (Hauri and Bucher, 1986) or casein are widely used and appear to be highly effective, with blocking of nitrocellulose being completed in 1 h at room temperature using Dulbecco’s phosphate buffered saline ‘A’ (137 mM sodium chloride, 2.7 mM potassium chloride, 8.1 mM.disodium hydrogen orthophosphate, 0.15 mM potassium dihydrogen orthophosphate, pH 7.3) containing 1% (w/v) casein (Harper et al., 1986). Blocking of nylon membranes requires far longer incubations and elevated temperatures, typically let-12 h at 60°C (Gershoni and Palade, 1982), and is one of the major problems with these membranes.

Uses After blotting, the proteins are present in an accessible state, bound to the solid matrix of the membrane. Early suggestions that this be used to facilite autoradiographic visualisation (Bumette, 1981; Towbin et al., 1979) did not prove popular owing to the variation in transfer efficiency of different proteins (Erickson et al., 1982). However, fluorographic procedures for use with membranes using salicylic acid have been developed (Luther and Lego, 1989), and some commercial water-based fluors are also suitable for this purpose. Membrane-bound proteins may also be assayed for enzymic function (Kanellis et al., 1989; Van den Berg, 1986). chemical reactivity (Contor et al., 1987; Gershoni et al., 1985a), or amino acid sequence (Kennedy et al., 1988). In sequencing applications, Immobilon membranes are frequently used due to their chemical properties (Shively et al., 1989). In addition, proteins thus extracted from the gel matrix may be used as immunogens (Abou-Zeid et al., 1987; Knudsen, 1985) or eluted from the membrane in a form of preparative electrophoresis (Parekh et al., 1985; Salinovi~h and Montelaro, 1986; Szewczyk and Summers, 1988). However, protein blotting is most often followed by reaction of the bound proteins with antibodies, prior to detection with antibody-specific labelled probes.

31

Immunodetection The use of antisera as probes allows the identification of the membmnebound proteins, and subsequent detection of bound antibody with a specific probe in a two-stage method considerably enhances sensitivity. While membranebound proteins may be detected with .directly la~lled.~tib~ies, this greatly reduces sensitivity (Bernstein et al., 1987), and is often not feasible; for example when assaying serum antibody in a clinical setting. If desired, antigen-bearing strips may be stored at -20°C for extended periods prior to immunoreaction (Bernstein et al., 1983, or may be treated after immunoreactionto remove bound antibodies, allowing re-use (Erickson et al., 1982; Legocki and Verma, 1981). The second stage probe is required to bind specifically to ‘immunoglobulin, but this requirement has been filled in many ways. Initially; staphylococcal protein A was used (Bumette, l981; Towbin, 1979). This has the property of binding,most types of IgG, along with some IgM (Goding, 1984). It is widely available, well characterised, and inexpensive; consequently, it is still widely used. How&er;.in many cases, it is insufficiently specific in its binding properties; this is particularly true when differentiation. of immunoglobulin classes, is required. More specific non-immunological binding proteins are currently available from commercial suppliers, such as the IgG-binding streptococcal protein G, but in most cases specific antibodies directed against the immunoglobulin of interest are used (Harper et al., 1988; Landini et al., 1985; Porath et al., 1,987). Labelling of the second stage probe may use radioisotopic or chromogenic principles. For the former, “‘1 is almost universally used, as it gives ‘.a% high intensity signal, thereby enhancing sensitivity. The range of chromogenic labels is somewhat wider. The most commonly used system is to label the probe with an enzyme and use precipitation of a coloured reaction product to locate bound antibody, These systems are frequently derived from histochemical stains; a typical example is the use of horseradish peroxidase-conjugated enzyme, combined with a substrate such as 4-chloro- 1-naphthol, which gives purple bands (Bers and Garfin, 1985; Nakane, 1968). The sensitivity of such a. system.can be further enhanced by the addition of soluble enzyme/antibody complexes which bind to the conjugated enzyme, thus increasing the amount of bound enzyme, and producing stronger bands (Bernstein et al., 1987; Stemberger et al., 1970). While autoradiographic procedures require time for the development of the image, typically from a few days to several. weeks, enzyme-labelled probes permit visualisation of the image within minutes. Against this, radiographic procedures permit re-exposure of the immunoblot in order to optimise band intensi~, while enzyme ,labelling is more of a “one-shot’ procedure. Direct visualisation *of bound probe using.attached gold particles is also available (Perides’et al.; 1986), although this appears more suited, to screening applications where. simplicity of the technique is paramount. It is also possible to use different: chromogenic labels in order to visual&e ,different antigens on the same membrane (Lin and Pagano, 1986), but this technique has’not yet become widely established. A novel variation on the use of chromogenic labelling is the use of .a luminescent reaction, allowing

32

the detection of bands on X-ray film as with radiola~lled probe. This allows re-exposure as detailed above, but has the time advantages of a chromogenic system, since the exposure time is typically less than a minute (Leong et al., 1986; Vachereau, 1989). However, the method requires careful optimisation, since the band pattern obtained changes with time (Harper and Murphy, in press). Luminescent systems are also available commercially (Amersham; New Brunswick Scientific), using chemically enhanced luminescent reactions.

The effects of protein denaturation Western blotting is a powerful analytical technique capable of the simultaneous identification of multiple immunogenic proteins, but as a consequence of the techniques involved, these proteins are in a highly denatured state. This is likely to prevent the reaction of the majority of antibodies present, allowing only those which react with conformation-insensitive epitopes to bind (Chapsal and Pereira, 1988; Grose and Litwin, 1988). This is particularly relevant for monoclonal antibodies; a polyclonal antiserum will contain antibodies to multiple epitopes on a protein, some of which are likely to be denaturation-resistant, but a monoclonal antibody will only be reactive against one epitope, and if this is rendered inactive then all immunoreactivity will be lost. It is commonly found that monoclonal antibodies fail to react in Western blotting. Even with polyclonal sera, an individual protein may be poorly reactive; for example, it has been shown that Western blotting is a poor detection system for the gpII1 glycoprotein of varicella-zoster virus, even with highly reactive sera (Dubey et al., 1989, Grose and Litwin, 1988). Consequently, it is important that failure to detect antibodies against a particular protein by Western blotting should not be regarded as indicating that no such antibodies are present. The presence of a high proportion of denaturation-sensitive antibodies in the early stages of the serological response may also explain the relatively slow appearance of reactivity to varicella-zoster virus with Western blotting of patient sera taken after primary infection (Harper et al., 1988). The Laemmli (1970) system of SDS-PAGE is commonly used prior to electroblotting, and is popular due to its high resolving power, which derives at least in part from the highly denatured state of the electrophoresed proteins. Modifications of the gel system aimed at repining more of the native protein structure have reported increased immunoreactivity when sulphydryl reagents are not used, allowing disulphide bridges to remain intact, and reduction or omission of SDS has also been reported to give increased levels of immunoreactivity (Birk and Koepsell, 1987; Cohen et al., 1986; Fehniger et al., 1984; Lammle et al., 1986; Samson et al., 1988). Since these modifications rely on reducing the denaturation of proteins during ele~trophoretic separation, care must be taken in their use. Omission of sutphydryl reagent can result in failure to solubilise aggregated proteins, giving poor solubility or the resolution of composite bands (Samson, 1986). Cohen et al. (1986), while retaining the normal level of SDS in their poly-

33

acrylamide gels (0.1%), reduced the concentration of SDS in their sample buffer from the 2% of Laemmli (1970) to O.l%, and reported improved detection of conformation-sensitive epitopes. However, they noted that this could adversely affect the sensitivity of the detection system in some cases, and that further reductions resulted in a loss of resolution. The presence of SDS during electroblotting has been reported to reduce subsequent immunoreactivity (Birk and Koepsell, 1987), while the presence of the zwitterionic detergent Empigen BB has been reported to restore some immunoreactivity (Christie et al., 1988). It appears that some limited renaturation of memb~e-Lund proteins can also occur during the blocking incubation, although the extent of this appears to vary with the blocking solution used (Birk and Koepsell, 1987; Ham-i and Bucher, 1986). The power of Western blotting lies in its ability to provide simultaneous resolution of multiple immunogenic proteins within a sample. In this, it differs from the majority of immunoassay techniques, and this has made it a highly valuable technique, with an eno~ous range of reported applications. While there are problems associated with the technique, it is not unique in this. Radioimmunoprecipitation. (RIP) represents the main alternative to Western blotting, and allows analysis of antigens in a far less denatured state. However, this method requires radiolabelling of the test antigen, and can falsely detect nonimmunogenic proteins by co-precipitation with reactive proteins. RIP can also fail to detect poorly soluble proteins which are easily detected by Western blotting (Harper and Grose, 1989). Ideally, when attempting to provide a complete analysis of the range of reactivities of antibodies, more than one technique should be used. If this is not done, the results obtained must be interpreted with due consideration given the limitations of the technique used. Applications

The antibody used in the initial immunoreaction may be of either known or unknown specificity. In the former case, monoclonal antibodies or sera of known specificity are used to assay unknown proteins. This technique provides a sensitive assay for the presence of immuno~active proteins, and has many other applications, for example in the analysis of cross-reacting epitopes with related viruses (Snowden and Halliburton, 1985). When antibodies of unknown specificity are used, they may be monoclonal, being assayed to determine the nature of their target immunogen, or a patient serum, being assayed for antibody against a particular pathogen. In particular, Western blotting is widely used to assay for the presence of antibodies against the human immunodeficiency virus (HIV) (Esteban et al., 1985). In this role, Western blotting has been shown to be more sensitive than some other forms of immunoassay (Griffith et al., 1989; Saah et al., 1987), although it is also prone to variation as a result of minor alterations in technique (Carrow et al., 1987). In addition, the interpretation of equivocal or borderline results obtained by Western blotting is more dif~cult than

34

with assays which give a numerical result, for which precise cutoff values may be set. Analysis of Western blots by densitometry rather than visually may help to counter this problem (Schiavini et al., 1989; Schmidt et al., 1987), but care must be taken to eliminate the effects of varying background staining along the antigen-bearing strip. Due to the ability of Western blotting to differentiate antibody binding by the molecular weight of the reactive protein, it allows elimination of many problems associated with non-specific binding in immunoassays. Despite several reports of false positive results when testing for HIV antibodies (Biberfeld et al., 1986; Courouce et al., 1986; Saag and Britz, 1986; Settergren et al., 1989; Thorpe et al., 1986, Van der Poe1 et al., 1986), and an apparently poor correlation between weakly positive or equivocal Western blot results and the development of HIV infection (Josephson et al., 1989; Lantin et al., 1989), Western blotting has become a widely-used reference technique for the establishment of antibody status to HIV (Centers for Disease Control, 1989). Refinements of the band patterns required to indicate a positive result, in particular a requirement for the simultaneous detection of multiple bands, appear to be effective in minimising false positive results (Centers for Disease Control, 1989; Zolla-Pazner et al., 1989), Western blotting has also been useful in analysing the development of the antibody response towards different HIV antigens (Lange et al., 1986), and in differentiating between HIV-l and HIV-2 (Ferroni et al., 1987; Werner et al., 1987). Other clinical applications of Western blotting are as a confirmatory test for HTLV retroviruses (Yamaguchi et al., 1988), and in the differentiation of primary and anamnestic (secondary) antibody responses in the herpesviruses (Harper et al., 1988). The particular advantages and disadvantages of Western blotting should be taken into account before using the technique, and must be considered when results are interpreted. Provided that it is optimised for the system in use, and work is undertaken in awareness of the limitations of the technique, Western blotting should continue to be highly valuable. A great deal of information has been obtained by its use, and the technique has undergone many changes since its inception. There is no reason to believe that this development will not continue.

Acknowledgements This work was supported by the Cancer Research Campaign (DRH, grant no. SP 1795) and the Joint Research Board of St. Bartholomew’s Hospital (KML).

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