High yield elution of proteins from sodium dodecyl sulfate polyacrylamide gels at the low-picomole level. Application to N-terminal sequencing of a scarce protein and to in-solution biological activity analysis of on-gel renatured proteins

被引:19
作者
CastellanosSerra, LR
FernandezPatron, C
Hardy, E
Santana, H
Huerta, V
机构
[1] Center for Genetic Engineering and Biotechnology,
来源
JOURNAL OF PROTEIN CHEMISTRY | 1997年 / 16卷 / 05期
关键词
SDS-PAGE; protein detection; protein micropurification; passive elution; trace enrichment;
D O I
10.1023/A:1026340923032
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-mu m 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 mu l for a 100-pmol BSA band), at room temperature. fluted 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.
引用
收藏
页码:415 / 419
页数:5
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