| pubmed-article:12236786 | pubmed:abstractText | Even for moderately sized proteins, the multiple occurrence of cysteine and lysine residues often prevents the specific labeling of polypeptides with a single probe. To increase specificity, a method was developed to convert the commonly available succinimidyl esters of fluorescent dyes into benzyl thioesters via trimethyl aluminum-activated benzyl mercaptan. The thioester can then be reacted very specifically with polypeptides containing an N-terminal cysteine residue, forming a stable amide bond, analogous to the native chemical ligation of peptide fragments. Both reaction steps are easy to perform and proceed to high yields. The practicability of the approach was demonstrated using the popular cyanine dye Cy5 and a soluble peptide, and it is expected to be applicable to a wide range of succinimidyl esters and both chemically and recombinantly synthesized proteins. The method should dramatically facilitate the preparation of proteins for experiments requiring exact positioning of labels, for instance, Förster resonance energy transfer studies. | lld:pubmed |
