Pappin, D. J., Coull, J. M., Koster, H. (May 1990) Solid-phase sequence analysis of proteins electroblotted or spotted onto polyvinylidene difluoride membranes. Anal Biochem, 187 (1). pp. 10-9. ISSN 0003-2697 (Print)0003-2697 (Linking)
Abstract
Electroblotted proteins noncovalently bound to polyvinylidene difluoride (PVDF) membranes are typically sequenced using adsorptive sequencer protocols (gas-phase or pulsed-liquid) that do not require a covalent linkage between protein and surface. We have developed simple chemical protocols where proteins are first electroblotted onto unmodified PVDF membranes, visualized with common protein stains, and then immobilized for solid-phase sequence analysis. Adsorbed, stained proteins are first treated with phenylisothiocyanate (PITC) to modify alpha and epsilon amines. The protein is then overlayed with a solution of 1,4-phenylene di-isothiocyanate (DITC), followed by a few microliters of a basic solution containing a poly(alkylamine). As the polymer dries onto the surface both polymer and remaining protein amino groups are crosslinked by DITC. The protein is thus immobilized to the membrane surface by entrapment in a thin polymer coating. The coating is transparent to the degradation chemistry, and extensive enough to remain immobilized even in the absence of any covalent link between polymer and surface. Partial modification with PITC allows for identification of N-terminal and internal lysine residues during sequencing. The process was tested with a variety of poly(alkylamines), linear and branched, with molecular weights ranging from 600 to over 100,000. Proteins bound in this manner were successfully sequenced using covalent (solid-phase) sequencer protocols with cycle times as short as 26 min.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | Absorption Amino Acid Sequence Electrophoresis, Polyacrylamide Gel/ methods Immunoblotting/ methods Isothiocyanates Lactoglobulins Membranes, Artificial Myoglobin Polyvinyls Protein Binding Thiocyanates |
| Subjects: | Investigative techniques and equipment bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification biotechnology > chromatography > protein purification |
| CSHL Authors: | |
| Communities: | CSHL labs > Pappin lab |
| Depositing User: | Matt Covey |
| Date: | 15 May 1990 |
| Date Deposited: | 11 Sep 2014 14:39 |
| Last Modified: | 11 Sep 2014 14:39 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/30782 |
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