Enzymatic cleavage and HPLC peptide mapping of proteins.
Detailed procedures are described for successfully digesting reasonably small quantities (i.e., usually > 10 pmol) of proteins with a variety of proteases and for then isolating the resulting peptides by reverse-phase HPLC. Since sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) appears to be the current method of choice for final purification of proteins for structural analysis, special attention is given to carrying out in-gel proteolytic digests on SDS-PAGE-separated proteins that have usually been stained with Coomassie Blue. A compilation of data from nearly 200 "unknown" samples is used to help provide realistic expectations with respect to the results that are likely to be obtained from carrying out in-gel proteolytic digests on large numbers of proteins.
Williams
K R
KR
W. M. Keck Foundation Biotechnology Resource Laboratory, New Haven, CT, USA. kenneth.williams@yale.edu
Stone
K L
KL
D000596:Amino Acids / Q000032:analysis; D001671:Biochemistry / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002918:Chymotrypsin / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D010434:Pepsin A / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D011506:Proteins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase; D014357:Trypsin; C036813:lysyl endopeptidase; D010434:Pepsin A