ISSN:
1573-4943
Keywords:
SDS-PAGE
;
protein detection
;
protein micropurification
;
passive elution
;
trace enrichment
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
Abstract A simple, reliable procedure for practically quantitative (90–98%) and fast (〈30 min) elution of proteins from SDS-PA gels is described with reproducible recoveries in the range from 100 to 1 pmol per band, which does not require the inclusion of detergents in the elution buffer. It consists in the combination of (1) highly sensitive on-gel protein detection (50 mol per band) with imidazole-SDS-zinc (reverse staining), (2) crushing of the protein band to produce 32-μm gel particles, and (3) vortexing of the slurry in a solution of a zinc-complexing agent, e.g. glycine 0.5 M or EDTA 100 mM (100 μl for a 100-pmol BSA band), at room temperature. Eluted proteins can be directly analyzed by RP-HPLC, quantitatively loaded onto a PVDF membrane, or, provided that they are previously renatured on-gel, analyzed by biological activity tests. The application of the procedure to in-solution enrichment of scarce proteins for N-terminal analysis is shown.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1026340923032
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