Strategies for internal amino acid sequence analysis of proteins separated by polyacrylamide gel electrophoresis.
Strategies for internal amino acid sequence analysis of proteins separated by polyacrylamide gel electrophoresis.
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通过聚丙烯酰胺凝胶电泳分离的蛋白质的内部氨基酸序列分析策略。
DOI：
10.1016/0021-9673(90)85148-o
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
R. Simpson
中科院分区：
文献类型：
--
作者：
L. D. Ward;G. E. Reid;R. Moritz;R. Simpson
An evaluation has been made of various stategies for obtaining internal amino acid sequence data from electrophoretically separated proteins. Electroblotting,in situproteolysis and extraction, and direct electroelution are compared. Electroblotting of protein or peptides from gels resulted in poor yields (typically, 1–7%). However, higher yields (3–67%) were achieved byin situenzymatic cleavage followed by acid extraction of the peptides from the gel. Peptides extracted from the gel were separated by reversed-phase high-performance liquid chromatography (RP-HPLC), on short, small-bore columns (100 × 2.1 mm I.D.), to enable recovery of peptides in small volumes (ca.50 μl) suitable for microsequence analysis. Capillary zone electrophoresis under acidic conditions (pH 2.5) was used to assess peptide purity before sequence analysis. Cysteine residues were identified in unmodified proteins or peptides by a characteristic phenylthiohydantoin (PTH)-amino acid derivative during sequence analysis. This derivative does not co-chromatograph with any known PTH-amino acid.Direct electrophoretic elution of protein from gels yielded between 45–50% of applied protein. Proteins recovered from gels by electrophoretic elution required further purification by inverse-gradient RP-HPLC [R. J. Simpson, R. L. Moritz, E. C. Nice and B. Grego,Eur. J. Biochem., 165 (1987) 21] to remove sodium dodecylsulphate and acrylamide-related contaminants for sequence analysis.
影响因子：
2.9
作者：
FLING, SP;GREGERSON, DS
通讯作者：
GREGERSON, DS
DOI：
10.1016/s0021-9258(18)61070-1
发表时间：
1987-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
P. Matsudaira
通讯作者：
P. Matsudaira
影响因子：
2.9
作者：
Xu,QY;Shively,JE
通讯作者：
Shively,JE