Abstract

A simple method for proteolytic digestion of Coomassie blue-stained proteins in a polyacrylamide matrix is presented. It consists of first reducing and alkylating the stained proteins with dithiothreitol and iodoacetamide in the presence of 0.1% sodium dodecyl sulfate and subsequent digestion with the endoproteinase LysC. The reduction and alkylation step was introduced since experiments with lysozyme and ribonuclease A showed that extremely complex peptide patterns were obtained if no precautions were taken to suppress disulfide bond formation during in-gel digestion of proteins. The advantage of this method is that no blotting step is required for generating internal sequences and that extensive proteolysis occurs which closely resembles that resulting from solution digests. The method has been successfully used to generate internal sequence data from low microgram quantities of proteins excised from 2-dimensional Coomassie blue-stained gels.

Keywords

IodoacetamideChemistryDithiothreitolPolyacrylamideChromatographyCoomassie Brilliant BlueProteolysisLysozymePolyacrylamide gel electrophoresisSodium dodecyl sulfateBiochemistryRibonucleaseTrypsinDigestion (alchemy)CysteineStainingBiologyEnzyme

MeSH Terms

Acrylic ResinsAlkylationAmino Acid SequenceMolecular Sequence DataOxidation-ReductionPeptide Mapping

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Publication Info

Year
1995
Type
article
Volume
224
Issue
1
Pages
75-82
Citations
201
Access
Closed

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Cite This

Paul Jenö, Thierry Mini, Suzette Moes et al. (1995). Internal Sequences from Proteins Digested in Polyacrylamide Gels. Analytical Biochemistry , 224 (1) , 75-82. https://doi.org/10.1006/abio.1995.1010

Identifiers

DOI
10.1006/abio.1995.1010
PMID
7710119

Data Quality

Data completeness: 81%