ANALYTICAL BIOCHEMISTRY 131, l-15 (1983) REVIEW Protein Blotting: Principles and Applications JONATHAN M. GERSHONI AND GEORGE E. PALADE Yale University, School of Medicine. 333 Cedar Street. Post Qjice Box 3333. New Haven. Connecticut 06510 KEY WORDS: protein blotting; nitrocelluiose: membrane filters: immune assays; Zetabind: DBM paper. Twenty-eight years ago, Smithies demon- strated that a starch gel could serve as a mo- lecular sieve through which zone electropho- resis of proteins could be carried out (95). Since then, repeated innovations and im- provements have brought the technique of protein separation by gel electrophoresis to the state we know today. The introduction of polyacrylamide gels (8 1) and of discontinuous buffer systems (20,75), the use of SDS’ to sol- ubilize and disaggregate protein complexes prior to electrophoresis (102), and the addition of SDS to discontinuous buffer systems (59,69) have all been major contributions to the development of this procedure, now one of the most widely used analytical and pre- parative tools in cellular and molecular bi- ology. Problems in gel electrophoresis. Initially, the main objective of these techniques was to monitor visually the homogeneity or hetero- geneity of a protein preparation and to follow the appearance or disappearance of a partic- ular “band” throughout a given experimental procedure. One-dimensional gels were found to be quite adequate when relatively simple protein samples, e.g., viruses ( 102), bacterio- phages (59,100) erythrocyte ghost mem- branes (25), were analyzed. But, as more com- plex systems demanded greater resolving I Abbreviations used: BSA; bovine serum albumin, DBM; diazobenzyloxymethyl, NC; nitrocellulose. PBS: phosphate-buffered saline. SDS; sodium dodecyl sulfate, ZB; Zetabind. power, new two-dimensional gel systems were developed (56,67). Such systems have been progressively improved to the point that they currently allow the efficient resolution of hundreds or even thousands of polypeptides found in some proteinaceous samples now under investigation (5,72). However, the task of unequivocally correlating a “band” or “spot” with a known function has often been difficult, especially when the resolution of the proteins has depended on their denaturation. Nevertheless, many approaches which allow the identification of a specific enzyme (45,53,64,92), antigen (1,16,73.91,101), gly- coprotein ( 16,4 1,42,88), hormone receptor (63,76,78,87,1 I3), or other protein-protein interactions ( 17,36,96) in a gel have been de- veloped. These techniques rely on maintain- ing at least one of the following prerequisites: (i) retention of the polypeptide’s specific ac- tivity throughout electrophoresis, (ii) possible renaturation of a denatured polypeptide, and (iii) covalent crosslinkage of the protein in question to a detectable ligand prior to elec- trophoresis. In these and other procedures, the actual processing of the gels entails multiple manipulations, including extensive incuba- tions and repeated washes. This is a time-con- suming operation, quite often prone to han- dling accidents, e.g., breakage and tearing of wet gels or cracking of drying gels. In addi- tion, the identification of the resolved poly- peptide bands generally requires relatively large amounts of reagents (e.g., antibodies) of- 1 0003-2697/83 $3.00 Cop)rght 6’ 1981 hv Academx Press. Inc All rtghtl of rrproductw, ,n any lorm rcsrivcd.