ABSTRACT
Large proteins do not penetrate gels for polyacrylamide gel electrophoresis (PAGE), and small proteins and peptides cannot be fi xed by blotting for detection or measurement. Even molecular weight markers do not transfer reliably, but their transfer is inversely dependent on molecular size. 1 We were driven to develop an alternative to
PAGE and Western blotting because fi brinogen and its multimeric derivatives, the proteins of principal interest to us, do not blot-transfer at all. By example, studies using Western blotting failed to detect all but degraded forms of fi brin(ogen), while our studies using direct immunoprobing showed that degraded fi brinogen comprise a miniscule percentage of the total (Figure 25.1) and intact fi brinogen and its crosslinked polymers comprise nearly all of the deposits. 2 The intact fi brinogen and its oligomers remain in the poyacrylamide gels like highly insoluble, precipitated blocks of coal as they separate from the SDS. Our solution was to develop an agarose-based derivative with acetaldehyde substituent groups that interact too weakly with amino groups to interfere with electrophoresis but could covalently link the protein and peptide to the gel matrix as alkyl amine adducts by simply immersing the gel in buffer containing cyanoborohydride to catalyze reductive amination of the aldehyde substituent groups. 3,4 Further, the gels could be used for multiple immunoprobing for tricolor staining of fi brinogen α, β, and γ chains 5 and enhancing sensitivity because retained antibody can be fi xed in-place for multiple processing without loss. The advantages are illustrated by their utility for detecting numerous fi brin(ogen) derivatives not known before 2,6,7,8 and detection of small peptides such as heme-octapeptide that cannot be fi xed by conventional fi xatives. 4 Even the usual molecular weight standards have variable blot-transfer characteristics, varying inversely with molecular size. 1 However,
FIGURE 25.1 Glyoxyl agarose electropherograms of saline (SAL) and SDS-urea (SDS-U) extracts of three atherosclerotic aortic intimas probed directly with anti-fi brinogen antibody showing only intact fi brinogen and oligomers, and virtually no degraded forms. The two lanes at the right (designated PL) are of a normal plasma sample run at indicated dilutions. Prior studies using Western blotting uncovered only degraded forms from intimal extracts because intact fi brinogen does not blot transfer. 2 Likewise, reduced gels failed to reveal degraded polypeptide chains of fi brinogen, only intact chains and cross-linked high molecular weight oligomers, which also do not blot-transfer well at all.
