In vitro labeling of proteins by reductive methylation: Application to proteins involved in supramolecular structures

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Abstract
Actin and tropomyosin, purified from both muscle and brain, and α-actinin, purified from muscle, have been labeled in vitro by reductive methylation to specific activities of greater than 105 dpm/μg protein. Actin so modified bound DNase I and polymerized identically to unmodified actin. Furthermore, the spectral properties of actin did not change after labeling. The interactions of labeled tropomyosin and α-actinin with F-actin were nearly identical to those of the unmodified proteins. These modified proteins comigrated with their unmodified counterparts in both SDS-containing polyacrylamide gels and isoelectric focusing gels. The labeled actin was quantitatively extracted from SDS-containing polyacrylamide gels (yield > 98% of radioactivity applied demonstrating that all of the radioactivity was protein bound. The reductive methylation procedure worked well at pH 8.0–8.5 in either pyrophosphate buffer or Bicine buffer using formaldehyde with [3H]-sodium borohydride as the reducing agent. The procedure could also be performed at pH 7.0 in phosphate buffer using [14C]-formaldehyde with sodium cyanoborohydride as the reducing agent. Proteins so labeled are ideal for use in quantitative experiments involving protein-protein interactions.