34 protein blotting

34

Protein Blotting

Principles and Applications

Peter FL Shewry and Roger J. Fido

1. Introduction Protein blotting was mtttally introduced m the late 1970s to identify protein antigens

that bound to spectfic antibodies (I,2) In this procedure, a complex protein fraction was separated by electrophorests and the proteins transferred and bound to a membrane, which was then probed with a radtolabeled antibody This concept and procedure were based on blotting and hybrtdrzatton methods developed for DNA by Southern (3) (and logrcally called Southern blottmg), which was subsequently modified for RNA (4) (less logtcally called Northern blotting by some authors). It is, therefore, not sur- prising that the protem/antibody procedure was called Western blotting (2), and that Southwestern (protein/DNA) and Far Western (or West Western) (protem/protem) procedures have since been developed In addmon, rt 1s possible to apply proteins to the membrane m solutron (dot-blotting), or to transfer them directly from tissues (squash blots and tissue prints), whereas the development of mtcroscale protein sequencing and ammo-acrd analysts has allowed the use of blotting to purify and characterize mdr- vtdual components of highly complex mixtures

It 1s clear from this brief mtroductton that protein blotting 1s a highly versatile procedure with many actual and potential apphcattons. These are briefly discussed m this chapter, wrth particular reference to plant brochemrstry and molecular biology More detailed protocols for the mdtvtdual procedures are provided (5,6), or are available m the references and standard texts (7-9) as well as equipment suppliers (e g , Applied Btosystems, Bto-Rad, and so forth)

2. Western Blotting A range of options are avarlable for Western blottmg, m choice of membrane, trans-

fer procedure and protein vtsualrzatron

2.1. Choice of Membrane

The most commonly used membranes are based on mtrocellulose (NC), which may be pure or supported. The pore size 1s tmportant, and we would recommend a 0.45~pm

From Molecular Biomethods Handbook Edlted by R Rapley and J M Walker 0 Humana Press Inc , Totowa, NJ

435

436 Shewry and F/do

pore membrane for general use but a smaller pore size (0 2 urn) for proteins of M, below approx 20,000 The mam disadvantages of NC membranes are that they tend to be fragile and have low-bmdmg capacity In contrast, nylon membranes are much stron- ger than NC membranes and have up to SIX ttmes the protein bmdmg capactty, although these are not compatible with the most widely used protein stains (coomasste blue and amide black) They also give higher levels of nonspectftc bmdmg and hence require more effective blockmg before probtng (see Subheading 2.3.).

Perhaps the best available membranes, although also the most expensive, are those based on polyvmyhdene difluortde (PVDF, 10) These combme high-binding capacity with mechanical strength and are particularly suited for mtcrosequencmg (see Subheading 2.5.).

2.2. Protein Transfer

Four methods have been described to transfer protein from the electrophorettc gel, based on simple dtffUston, capillary action (similar to Southern’s origmal DNA-blottmg procedure), vacuum blotting, and electroblottmg. Of these, only electroblottmg 1s widely used today

Electroblottmg uses electroelutton to transfer protems from the gel to the membrane and generally gives more rapid and effective transfer than the other methods However, the rate and efficiency of transfer are affected by a number of factors, mcludmg the protem M, (large proteins transferring less efficiently) and isoelectric point (PI), the acrylamide concentration m the gel and the transfer buffer. We generally use a buffer contammg 20% (v/v) methanol and 0.2% (w/v) sodium dodecyl sulfate (SDS), which gives efficient transfer while preventing swellmg of the gel. However, for the transfer of larger size protems (M, >75,000), we use a nonmethanol Tns-borate buffer system (10)

A wide range of apparatus ts available commerctally, which falls mto two types: wet vertical tank systems and semtdry horizontal systems Although the latter have been reported to be cheaper (using less buffer) and more rapid to use, we obtain consistently good results with a wet tank system, reusing the buffer up to five times without loss of transfer efficiency

2.3. Total-Protein Detection

Stammg of membrane-bound protems can be used to confirm visually the efficiency and quality of transfer It is also used to determine the positions of molecular-weight marker proteins, although prestamed markers can also be used

Filters can be stained with standard stains used for gels, such as Coomasste brtlhant blue R250, amtdo black, India mk, Aurodye, and colloidal iron. In addition, rapid and reversible stammg prior to mnnunodetectton can be carried out either with Ponceau S or Amtdo black. This allows the proteins of interest or standards to be marked lightly with a pencil before destammg with Tris buffered salme contammg 0 05% (v/v) Tween 20 (TTBS).

Once protems have been bound to the filter they can be probed with the antibody to identify those that react. However, because the antibody is also a protem it will bmd nonspectfically to the filter unless all unused bmdmg sites are blocked. This can be achieved by using a cheap and widely available protein that will not react with the antibody of interest, such as bovme serum albumin (BSA) used at a concentration of up

Pro tern B/o ttmg 437

A Alkaltne Phosphatase Conjugated Primary

Primary Antlbody

Fig 1 Detection of bound proteins using labeled primary (A) and secondary (B) antibodies

to 5% (w/v). However, even BSA can be expensive when used m large amounts and an even cheaper source of blockmg protein is nonfat dried milk powder, of the “Marvel” type One blocking cocktail, called BLOTTO (II), consists of a 5% solution of low-fat dried milk m Tris-buffered salme containing anttfoam and antimicrobial agents BLOTTO, or 1% BSA, must also be used during the antibody binding, washing, and detection m order to mamtam the blocking action.

After blocking, the membrane can be hybridized with the antibody of interest (called the primary antibody). However, m order to visuahze the bound antibody, it is necessary to either label tt directly or to use a secondary detectton system. Labelmg of the primary antibody can be achieved by iodmation of tyrosme residues, with 1251, allowmg detection of the bound antibody by autoradiography Although highly sensitive, radtotodmatton 1s also very hazardous and use of a fluorescent label, such as fluorescem isothiocyanate, is safer

Although labeling of the primary antibody is still used m some studies, it is more usual to use a secondary detection system, either a second antibody or hgand (Fig. 1). A range of second antibodies are available to detect different types of primary antibodies (species and Ig isotypes) Thus, if the primary antibody is an IgG raised m rabbits, it 1s necessary to use an antirabbit IgG ratsed m a different species (for example, goat) as the second anttbody. This second antibody is itself coqugated to a detection system, allowing its presence to be detected. The most popular detection systems are conjugated enzymes, ather horseradish peroxidase (HRP) or alkalme phosphatase (AP) The addition of sub- strate results m the formatton of an msoluble, colored product at the bmdmg site, reveal- ing the bound antibody HRP was the first enzyme used for this purpose, but is relatively msensitive and the color fades on exposure to light Alkaline phosphatase 1s generally preferred because it is more sensitive and produces a stable end color when used with sub- strates, such as 5-bromo-4-chloro-3-mdolyl phosphate and mtro-blue tetrazolmm

Alternative secondary-detection systems are also available, notably the highly sensitive enhanced chemilummescence (ECL) system marketed by Amersham Interna- tional plc. In this system, an HRP-labeled second antibody 1s used to oxidize Lummol,

438 Shewry and Fide

to emit light that 1s enhanced approx 1 OOO-fold and detected using photographic film. Alternatively, the prrmary antibody can be reacted with Protein A, Protein G, or gold- labeled second antibody, and enhanced with silver (Bio-Rad)

The advantage of secondary-detection systems is enhancement of the signal, result- mg m higher sensmvity Thus, the use of Protein A, Protein G, or second antibody enhances the reaction of the primary antibody (Fig. l), whereas further enhancement is achieved by the ECL or silver-enhancement procedures

In most cases, mununoreactive protems can be readily identified by comparisons of stained gels wtth blots made from duplicate unstained gels However, there may be a problem m some cases, for example, wrth complex patterns of proteins on 2-D gels In this case, it may be necessary to confirm the identity by stammg filters for total protein after mnnunodetection which can be achieved by using 0 1% (v/v) India mk m TTBS (12), allowmg the chromogemcally stained immunoreactlve proteins to be identified

2.4. Applications of Western Blotting

The followmg examples are selected to illustrate the range of apphcattons of West- ern blotting m plant science

2 4 1. Analysrs of Transgemc Plants

The development of transformation systems for many plants, mcludmg major cereal, oilseed, and legume crops, provides an opportumty to mampulate aspects of growth, development, and composmon by genetic engineering. Western blotting 1s an impor- tant tool for such studies, m order to confirm the identity of the transgene product and to determme its pattern of expression

For example, scientists m our mstitute are transformmg wheat and mtordeum (a novel “hybrrd” cereal derived from combmmg the genomes of pasta wheat and a wild-barley species) wtth genes encoding high-mol-wt subunits of glutenm m order to determine the effects on the elasticity, and hence, breadmakmg quality of doughs This work has been facthtated by the availabihty of a monoclonal antibody (MAb) (IFRN 1601) that is highly specttic for a subgroup of high-mol-wt subunits (called x-type subunits), as shown m Fig. 2. There are numerous other examples of this apphcation, for example Higgins and Spencer (13) who studied the expression of vicilm protein m transgemc tobacco plants

2 4 2 Punficatlon of Antbodres by lmmunoaffin~ty Blottmg

Polyclonal antisera consist of mixtures of antibodies, mcludmg those that are specific for the protem used as anttgen It is possible to purify small amounts of specific antibodies by binding to protems which have been transferred to diazotized paper (14) or mtrocellulose (15) The antibody can then be released by pH shock with 0 2 Mglycme HCl buffer at pH 2 8, and the pH adjusted to 7 0 for storage These methods were mmally developed for studies of mammahan cytoskeletal protems, but we have since used them to purify polyclonal antibodies to plant protems for use m nnmunolocalization studies

2 4 3 ldentlftcatlon of Allergens

A number of plant proteins are major allergens, and can cause severe symptoms m sensitized mdivtduals The most widely studied are soybean and peanut, both of which contam a number of apparently unrelated food allergens Other food allergens include

Protein Blotting 439

Fig. 2. Expression of wheat high-mol-wt glutenin subunit lDx5 in seeds of transformed tritordeum line HT 28. (A) Total seed proteins separated by SDS-PAGE and stained with Coomassie BBR250. (J3) The proteins were transferred to nitrocellulose and detected with the MAb IFRN 1601, followed by second antibody labeled with alkaline phosphatase. M = Seeds of the wheat cv. M&&a, containing BMW subunit lDx5. T = Single seeds of tritordeum transformed with the wheat 1 Dx5 gene. C = seeds from plants of tritordeum that had been regenerated but not transformed. The high-mol-wt subunits are indicated by brackets and subunit lDx5 by arrowheads.

2s albumin seed storage proteins of Brazil nut, castor bean, and mustard, and a pro- teasekunylase inhibitor of rice. In addition, inhalation of flours of wheat and barley can result in an allergenic response known as baker’s asthma, and the active proteins have been identified as low-M, inhibitors of the cereal a-amylasekrypsin inhibitor family. Western blotting can be a powerful tool in the analysis of allergenic proteins, using IgE

440 Shewry and F/do

fractions from sera of sensitive mdividuals to probe protems extracted from plant materials that elicit a response (16,17)

2.4.4 Charactenzatlon of Ant/body Specrfiaty

There are two approaches to raising MAbs The first is to purify the protein of mter- est, and to use this to raise a library of MAbs. In this case, all of the MAbs should recognize the protein of interest, although they may well recognize different epitopes and differ m then specificity and titer. Alternatively, it is possible to nnmunogemze with a mixture of proteins and then characterize a larger number of antibodies, m order to identify those that are specific for mdivrdual proteins In both cases, Western blottmg can be used to determine antibody specificity.

For example, we have raised a hbrary of MAbs to a wheat glutemn fraction m order to identify clones that were specific for the different protein types present tn the mixture One such MAb, called IFRN067, proved to be of particular interest because its degree of bind- mg to gluten protein extracts from various gram samples was correlated with breadmakmg performance (r = 0.497) Western blotting of 1 -D SDS-polyacrylamide gel electrophoresis (PAGE) separations showed specific binding to a major band of M, about 45,000, but tt was not possible to identify the precise nnmunoreactrve proteins among the complex mixture This was finally established by double-stammg (nnmunodetection followed by India mk) (12) of a Western blot of proteins separated using a 2-D pollectic focusing (IEF)/SDS- PAGE system This allowed the nnmunoreactive band to be separated into two major components of similar M,, which were unequivocally identified m the protein mixture (Fig. 3)

2.4.5 Analysis of Plant Development

There are numerous examples of the use of protein blotting to study plant development and to show changes m the amount of mdividual proteins present m a complex mtxture Blotting can give much greater specificity and sensitivity than conventional protein stammg For example, we have used a wide specificity MAb (IFRN 0610) to study the amount and pattern of gluten-protein synthesis m developing caryopses of wheat (Fig. 4). This allows the detection of protems from only 7 d after anthesis, sev- eral days before they can be reliably detected by protem stammg

2.5. Microsequencing of Blotted Proteins

One of the most widely used applications of protein blotting is for microsequencmg, using automated pulsed-hqurd and gas-phase sequencers that can determine the N-terrmnal ammo sequences of 20 pmols or less of proteins. This ehmmates the need to purify protems by conventional methods and provrdes protein that is pure and easy to handle A range of filters based on glass fiber (chemically activated, sillcomzed or coated with quaternary ammomum polybases) were mmally used, but most workers currently pre- fer PVDF membranes, either Immobilon TM (Millipore Corp ) or Pro-BlotTM (Apphed Biosystems, Inc ) The latter has been specially developed for use m the Applied Biosystems Model 477A Pulsed Liquid Sequencer

2.6. Tissue Prints and Dot-Blots

The presence and location of protems in plant tissues can be determined using a modified Western-blottmg procedure called tissue prmtmg or squash blottmg The


Fig. 3. (A) Immunodetection with MAb IFRN067 and (B) double-staining procedure using imrnunodetection followed by total protein staining with India ink, of a Western blot of glutenin proteins separated using a 2-D IEF/SDS-PAGE system. This allowed the immunoreactive band to be separated into two major components of similar M,, which were identified (observed as purple spots [arrowed]) among the nonreactive spots within the protein mixture. Taken from Brett et al., 1992 (see ref. 18).

proteins are not extracted from the tissue, but transferred directly to a nitrocellulose membrane and then detected using an antibody and one of the labeling systems discussed previously. A surprisingly high level of resolution can be obtained if the protocol is optimized to suit the experimental material. A good example of tissue printing is provided by a recent study of invertase distribution in leaf tissue (19) (Fig. 5).- -

Dot-blotting is a simplified Western-blotting procedure, with the protein extract applied directly to the nitrocellulose membrane as a small spot. It has the advantage of being simple and rapid, and is reported to be approx lo- to lOOO-fold more sensitive than electroblotting. It is also possible to load dilute solutions as multiple loadings, with the membrane being allowed to dry in between. The speed and simplicity mean that it is readily adapted to automated or semi-automated analysis of multiple samples, and a number of systems are available commercially. For example, the Bio-Dot system from Bio-Rad has either 96 wells or 48 slots attached to a common manifold base to allow application and washing under vacuum. The membrane is processed and probed as for conventional Western blotting.

442 Shewty and Fido

Fig. 4. Use of a wide specificity MAb to study the amount and pattern of gluten-protein synthesis in developing caryopses of Chinese Spring wheat 5-40 d after flowering. (A) Total protein extracted on an equal weight basis and SDS-PAGE followed by (B) itnmunodetection of transferred protein by MAb IFRN 0610, as in Fig. 2. The groups of gluten proteins are indicated by brackets; 1, high-mol-wt subunits of glutenin; 2, w-gliadins; 3, low-mol-wt subunits of glutenin, a-type gliadins, and y-type gliadins.

3. Southwestern and Far-Western Blotting Southwestern blotting is a combination of protein and DNA blotting that was devel-

oped to identify DNA-binding proteins in crude protein extracts from nuclei (20). Such proteins could be involved in DNA replication or, by binding to regulatory sequences present in the 5’ upstream regions, the control of gene expression. The concept is simple. The crude-protein extract is initially separated by SDS-PAGE and transferred to a nitrocellulose membrane. It is then probed with a DNA sequence that is labeled either isotopically or using a nonradioactive system. Although Southwestern blotting is most widely used to identify proteins that bind to a characterized DNA sequence (20,21), it


B

Fig. 5. Tissue printing from peony (Paeonia o#kinaZis) leaves. (A) Protein transfer from a leaf from which the epidermis has been partially removed, revealed by amido-black staining. (B) Immunolocalization of acid-invertase to the vascular regions. Kindly provided by A. Kingston-Smith.

can also be used to select unknown DNA fragments that bind to characterized proteins, as a semipreparative system (22).

Far-Western (or West-Western blotting) is also derived from conventional Western blotting, but the blotted proteins are probed with a nonantibody protein in order to detect protein-protein interactions (23,24). The bound protein can then be detected using a specific antibody that uses a standard labeling system. Neither Southwestern blotting nor Far-Western blotting have so far been widely used in plant science.

Acknowledgments The authors are grateful to A. Tatham, P. Barcelo, F. Barro, and P. Lazzari (IACR-

Long Ashton Research Station) for providing Fig. 2. IACR receives grant-aided sup- port from the Biotechnology and Biological Sciences Research Council of the United Kingdom.

References 1. Towbin, S. A., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins

from polyacrylamide gels to nitrocellulose: procedure and some applications. Proc. Natl. Acad. Sci. USA 76,4350-4354.

2. Burnette, W. N. (1981) “Western Blotting”; electrophoretic transfer of proteins from sodium dodecylsulphate-polyacrylamide gels to unmodified nitrocellolose and radio- graphic detection with antibody and radioiodinated Protein A. Anal. Biochem. 112,195203.

3. Southern, E. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98,503-517.

4. Alwine, J. C., Kemp, D. J., and Stark, G. R. (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridisation with DNA probes. Proc. Natl. Acad. Sci. USA 74,5350-5354.

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Fide, R J , Tatham, A S and Shewry, P R (1993) Applications of protein blotting m plant biochemistry and molecular biology, m Methods in Plant Blochemutry, vol IO, Molecular Bzology (Bryant, J , ed ), Academic, London, pp 10 l-l 15 Fide, R J , Tatham, A S , and Shewry, P R (1995) Western blotting analysis, m Meth- ods In Molecular Biology, vol 49 Plant Gene Transfer and Expression Protocols (Jones, H , ed ), Humana, Totowa, NJ, pp 423-437 BJerrun, 0 J and Heegaard, N H H , eds (1988) Handbook of Zmmunoblottzng of Pro- telns Volume I, Technrcal Descrtptlons (265 pp ), Volume IZ, Expenmental and Clvucal AppEzcatzons (2 14 pp ), CRC, Boca Raton, FL Dunbar, B. S , ed (1994) Protem Blottzng A Practzcal Approach IRL, Oxford Umver- sity Press, Oxford, UK, 280 pp Electrophoresis (1993) Paper symposium-Protein Blottmg The Second Decade, vol 14, pp 829-960 Baker, C S , Dunn, M J , and Yacoub, M H (1991) Evaluation of membranes used for electroblottmg of proteins for direct automated rmcrosequencmg Electrophoreszs 12,342-348 Johnson, D A , Gautsch, J W , Sportsman, J R , and Elder, J H (1984) Improved tech- nique utihzmg nonfat dry milk for analysis of proteins and nucleic acids transferred to mtrocellulose Gene Anal Tech 1,3-8 Ono, T and Tuan, R S ( 1990) Double staining of unmunoblot using enzyme histochem- istry and mdia mk Anal Bzochem 187, 324-327 Higgms, T. J V and Spencer, D (1991) The expression of a chimeric cauliflower mosaic virus (CaMV-35S)-pea vicilm gene m tobacco Plant Scz 74,89-98 Olmsted, J B (198 1) Affinity purification of antibodies from diazotizes paper blots of heterogeneous protem samples J Bzol Chem 256, 11,955-l 1,957 Tailm, J C , Olmsted, J B , and Goldman, R D (1983) A rapid procedure for preparing fluorescem-labeled specific antibodies from whole antiserum its use m analysmg cytoskeletal architecture J Cell Bzol 97, 1277-l 282 Donovan, G R and Baldo, B A (1993) Immunoaffimty analysis of cross-reacting allergens by protein blotting Electrophoreszs 14,917-922 Sandiford, C. P , Tee, R D , and Newman-Taylor, A J (1995) Identification of crossreactmg wheat, rye, barley and soya flour allergens using sera from mdividuals with wheat-Induced asthma Clrn Exp Allergy 25,340-349 Brett, G M , Mills, E N C , Tatham, A S , Fide, R J Shewry, P R , and Morgan, M R A (1993) Immunochemical identitiction of LMW subunits of glutenm associated with bread-making quality of wheat flours Theor App Genet 86,442-448 Kingston-Smith, A H , and Pollock, C J (1996) Tissue level localization of acid mvertase m leaves an hypothesis for the regulation of carbon export New Phytol 134,423-432 Bowen, B , Stemberg, J , Laemmh, U K , and Wemtraub, H (1980) The detection of DNA-binding proteins by protein blotting Nucleic Aczds Res 8, l-20 Misklmms, W K , Roberts, M P , McClelland, A , and Ruddle, F H (1985) Use of a protein- blotting procedure and a specific DNA probe to identify nuclear proteins that recogmze the promoter region of the transfernn receptor gene Proc Nat1 Acad Scr USA 82,6741-6744 Keller, A D and Mamatis, T (1991) Selectron of sequences recogmzed by a DNA bmdmg protein using a preparative Southwestern blot Nucleic Aczds Res 19,4675-4680 Macgregor, P F , Abate, C , and Curran, T (1990) Direct cloning of leucme zipper proteins Jun bmds cooperatively to the CRE with CRE-BP1 Oncogene 5,45 1458 Grasser F A , Sauder, C , Haiss, P , Hille, A , Komg, S , Gottel, S , Kremmer, E , Lemenbach, H P , Zeppezauer, M , and Mueller-Lantzsch, N (1993) Immunological detection of proteins associated with the Epstein-Barr vrrus nuclear antigen 2A VzroZogy 195,550-560

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