Linked Life Data

8053543

Source:http://linkedlifedata.com/resource/pubmed/id/8053543

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1994-9-7
pubmed:abstractText
An improved and simplified procedure for enzymatic digestion of proteins bound to polyvinylidene difluoride (PVDF) membranes for obtaining internal protein sequence data is presented. This improved procedure is compatible with various enzymes (trypsin, endoproteinase Lys-C, endoproteinase Glu-C, and clostripain) and is performed in the presence of 1% hydrogenated Triton X-100 (RTX-100)/10% acetonitrile/100 mM Tris, pH 8.0, followed by microbore HPLC purification of the recovered peptides. Previously published techniques required treatment of the PVDF-bound protein with polyvinylpyrrolidine M(r) 40,000 (PVP-40) prior to digestion, in order to prevent adsorption of the enzyme to the membrane. Unfortunately, contaminants produced from residual PVP-40 interfere with subsequent peptide mapping. We have found that when RTX-100 is used in the digestion buffer, no pretreatment of the PVDF-bound protein with PVP-40 is necessary. Advantages of this improved (one-step) procedure over the two-step PVP-40 procedure are (a) the elimination of undesirable contaminants associated with PVP-40, (b) a decrease in the time required for the technique, and (c) a reduction in sample manipulation. Peptide maps and recoveries from PVDF-bound standard proteins (4 micrograms each) enzymatically digested with this one-step method are compared with those obtained from the standard PVP-40 method. In addition, peptide maps and internal sequence data from low-level quantities of unknown proteins enzymatically digested with the improved procedure are presented. To date, this improved, one-step procedure has been successfully applied to 52 PVDF-bound unknown proteins (0.7-10 micrograms) of varying molecular weight (19-300 kDa) for which internal sequence data were obtained.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0003-2697
pubmed:author
pubmed:issnType
Print
pubmed:volume
218
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
112-7
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1994
pubmed:articleTitle
An improved procedure for enzymatic digestion of polyvinylidene difluoride-bound proteins for internal sequence analysis.
pubmed:affiliation
Rockefeller University Protein Sequencing/Howard Hughes Medical Institute Biopolymer Facilities, New York, New York 10021.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.