Isoelectric focusing of tryptic peptides from hemoglobin subunits by immobilized pH gradients

The technique of isoelectric focusing on immobilized pH gradients (IPG) has been applied to the analysis of tryptic digests of alpha- and beta-chains of human hemoglobin. Using peptides purified by RP-HPLC as a reference, it was possible to create a peptide map in the single IEF dimension. Unfortunately, it was not possible to find experimental conditions (medium for migration and staining) which would allow the detection of peptides of less than 10-12 residues. Almost all the bands visible on the gel could be assigned to known peptides. In order to obtain these results the IPG runs were performed in 8 M urea containing 0.5% carrier ampholytes and the gel stained with colloidal Coomassie brilliant blue G-250, in the presence of a high-salt concentration and at acidic pH.