Vera J C, Rivas C I, Cortés P A, Cárcamo J O, Delgado J
Instituto de Bioquímica, Universidad Austral de Chile, Valdivia.
Anal Biochem. 1988 Oct;174(1):38-45. doi: 10.1016/0003-2697(88)90516-7.
Proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were stained in situ with either 5-(dimethylamino)-1-naphthalene sulfonyl chloride (dansyl chloride) or fluorescein isothiocyanate. This staining procedure can be carried out in less than 30 min without previous fixation of the proteins. It is not dependent on such factors as charge or molecular weight of the proteins and can detect 50 ng of protein in a 10-mm-wide gel slot. Fluorescent staining with dansyl chloride was used to localize proteins after electrophoresis for subsequent electroelution, amino terminal analysis, and peptide mapping. The electroelution can be carried out in less than 3 h with yields approaching 100%. The staining of only one strip of a preparative gel allowed the electroelution of proteins without covalent modification. For amino terminal analysis, identical results were obtained when the hydrolysis step was carried out after electroelution or directly in the gel pieces. The peptide mapping can be carried out with the proteins in solution (after electroelution) or directly in the gel pieces. The amino terminal and peptide mapping analysis of each protein in a mixture can be completed within 30 h from the beginning of the electrophoretic fractionation. The method appears to be applicable to a wide range of proteins showing very different biochemical properties.
通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分离的蛋白质，用5 - （二甲基氨基） - 1 - 萘磺酰氯（丹磺酰氯）或异硫氰酸荧光素进行原位染色。这种染色程序可在不到30分钟内完成，无需事先固定蛋白质。它不依赖于蛋白质的电荷或分子量等因素，并且可以在10毫米宽的凝胶槽中检测到50纳克蛋白质。用丹磺酰氯进行荧光染色用于电泳后定位蛋白质，以便后续进行电洗脱、氨基末端分析和肽图谱分析。电洗脱可在不到3小时内完成，产率接近100%。仅对制备凝胶的一条带进行染色就可以在不进行共价修饰的情况下电洗脱蛋白质。对于氨基末端分析，在电洗脱后或直接在凝胶片中进行水解步骤时，可获得相同的结果。肽图谱分析可以用溶液中的蛋白质（电洗脱后）或直接在凝胶片中进行。从电泳分离开始，混合物中每种蛋白质的氨基末端和肽图谱分析可在30小时内完成。该方法似乎适用于具有非常不同生化特性的广泛蛋白质。
