Reutilization of Immunoblots after Chemiluminescent Detection

✍ Scribed by Scott H. Kaufmann

• Book ID: 102560207
• Publisher: Elsevier Science
• Year: 2001
• Tongue: English
• Weight: 101 KB
• Volume: 296
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2001.5313

✦ Synopsis

does not have any noticeable impact on the recovery of tryptic peptides.

We also observed that relative concentration of all peptides in the digests of silver-stained bands, which were directly treated with trypsin (i.e. washing steps as well as destaining, reduction and alkylation were omitted), was dramatically lower. Nevertheless, the number of detected peptide was always sufficient for unambiguous identification of BSA upon searching a database. We therefore speculate that the sequence coverage of MALDI peptide maps alone does not constitute an adequate measure of the digestion efficiency and should be complemented by direct quantification of peptide products.

Thus we have demonstrated that application of isotopically labeled peptide standards and MALDI MS enabled direct and quantitative evaluation of the efficiency of in-gel digestion. The method paves the way for further optimization of sample processing routines, thus improving sensitivity and throughput of the characterization of proteomes by mass spectrometry.

Acknowledgments. The authors are grateful for members of Protein and Peptide Group for experimental support and useful discussions.