Carrey E A, Hardie D G
Anal Biochem. 1986 Nov 1;158(2):431-5. doi: 10.1016/0003-2697(86)90571-3.
Sections of nitrocellulose containing bound 32P-labeled polypeptides were excised from "Western" blots and exhaustively digested by trypsin in order to analyze the distribution of phosphorylation sites between the products of limited proteolysis of the multifunctional protein CAD. Using the criterion of analytical isoelectric focusing, the 32P-peptides obtained by this method were found to be similar, although not identical, to peptides obtained by a more conventional digestion of trichloroacetic acid precipitates. Digestion on Western blots is more straightforward than electrophoretic elution of individual gel slices, gives better recoveries than direct digestion of gel slices, and is particularly suitable for peptide mapping of small peptides which bind to nitrocellulose but would diffuse out of polyacrylamide gels during the commonly used fixing and staining procedures.
从“蛋白质免疫印迹法”中切下含有结合了32P标记多肽的硝酸纤维素膜条，并使用胰蛋白酶进行彻底消化，以分析多功能蛋白CAD有限蛋白酶解产物之间磷酸化位点的分布。使用分析性等电聚焦标准，发现通过该方法获得的32P肽与通过更传统的三氯乙酸沉淀消化获得的肽相似，尽管不完全相同。在蛋白质免疫印迹法上进行消化比单个凝胶切片的电泳洗脱更直接，回收率比凝胶切片的直接消化更好，并且特别适用于与硝酸纤维素结合但在常用的固定和染色程序中会从聚丙烯酰胺凝胶中扩散出来的小肽的肽图谱分析。
