Transfer of proteins to membranes facilitates both cyanogen bromide cleavage and two-dimensional proteolytic mapping.
We have found that the transfer of gel-fractionated proteins to membranes facilitates phosphopeptide mapping. Nitrocellulose proves to be an excellent matrix for both cyanogen bromide cleavage and proteolytic digestion. Digestion of p56lck bound to a nitrocellulose membrane with cyanogen bromide or trypsin generated patterns of phosphopeptides indistinguishable from those produced by digestion of p56lck eluted from a gel. Immobilon-P and nylon membranes can also be used for proteolytic mapping, but not for cyanogen bromide cleavage. Since the use of membrane-bound protein eliminates the need for elution and precipitation of the protein, analysis is rapid. In addition, the recovery of the peptides from proteins digested on membranes is better and more consistent than it is from eluted and precipitated proteins.
Luo
K
K
Molecular Biology and Virology Laboratory, Salk Institute, San Diego, California 92138.
Hurley
T R
TR
Sefton
B M
BM
D000818:Animals; D003101:Collodion / Q000378:metabolism; D003488:Cyanogen Bromide; D004338:Drug Combinations; D005048:Etorphine; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008567:Membranes, Artificial; D008728:Methotrimeprazine; D051379:Mice; D009757:Nylons; D010449:Peptide Mapping / Q000379:methods*; D011145:Polyvinyls; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D013945:Thymoma / Q000378:metabolism / Q000473:pathology; D013953:Thymus Neoplasms / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D014357:Trypsin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000473:pathology / Q000648:ultrastructure; D005465:Fluorocarbon Polymers
D003101:Collodion; D003488:Cyanogen Bromide; D004338:Drug Combinations; D005048:Etorphine; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008567:Membranes, Artificial; D008728:Methotrimeprazine; D009757:Nylons; D011145:Polyvinyls; D011505:Protein-Tyrosine Kinases; D011506:Proteins; D014357:Trypsin; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C004820:Immobilon