S-CARBOXYMETHYLATION OF PROTEINS TRANSFERRED ONTO POLYVINYLIDENE DIFLUORIDE MEMBRANES FOLLOWED BY INSITU PROTEASE DIGESTION AND AMINO-ACID MICROSEQUENCING
S-CARBOXYMETHYLATION OF PROTEINS TRANSFERRED ONTO POLYVINYLIDENE DIFLUORIDE MEMBRANES FOLLOWED BY INSITU PROTEASE DIGESTION AND AMINO-ACID MICROSEQUENCING
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DOI：
10.1002/elps.1150130129
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发表时间：
1992-03-01
期刊：
影响因子：
2.9
通讯作者：
IWAMATSU, A
中科院分区：
文献类型：
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作者：
IWAMATSU, A
固定化蛋白质的S-羧甲基化，通过凝胶电泳分离和transblotted到聚偏二氟乙烯（PVDF）膜进行了描述。使用原位蛋白酶消化以高产率产生适合于微测序的肽片段。这些片段也可以用来产生肽图，通过反相高效液相色谱法（HPLC），表现出良好的重现性，并从digestrin在solution.This技术获得的结果相媲美，使它有可能确定半胱氨酸残基Edman降解过程中。仅使用50-200 pmol就可测定蛋白质中约70%的氨基酸残基。使用10-50 pmol的材料施加到聚丙烯酰胺凝胶上，获得用于产生寡核苷酸探针的足够信息。
S-Carboxymethylation of immobilized proteins, separated by gel electrophoresis and transblotted to polyvinylidene difluoride (PVDF) membranes is described. In situ protease digestion was used to produce peptide fragments in high yields that were suitable for microsequencing. These fragments could also be used to produce peptide maps, obtained by reverse-phase high performance liquid chromatography (HPLC), that exhibited excellent reproducibility and were comparable to results obtained from digestions in solution.This technique makes it possible to identify Cys residues during Edman degradation. Approximately 70% of the amino acid residues from a protein were determined using only 50-200 pmol. Sufficient information for production of oligonucleotide probes was obtained using 10-50 pmol of material applied to a polyacrylamide gel.