Hermansen L F, Pedersen O, Sletten K
Department of Biochemistry, University of Oslo, Norway.
Electrophoresis. 1993 Dec;14(12):1302-6. doi: 10.1002/elps.11501401199.
A protein transfer method which allows elution and immobilization of polypeptides onto a polyvinylidene difluoride (PVDF) membrane has been developed. The protein band in a gel is eluted by centrifugation. The centrifuge-blotting procedure involves the following steps: (i) visualization of the protein in a sodium dodecyl sulfate (SDS)-polyacrylamide gel with 1 M KCl, (ii) excision of the protein band and equilibration for 15 min in a solution of 0.05% SDS/5% methanol/0.02% dithiothreitol in distilled water, (iii) placing the gel piece in direct contact with the PVDF membrane in the receptacle, (iv) centrifugation at 3000 g for 1 h. A 10 kDa cut-off dialysis membrane is placed beneath the PVDF membrane to retain nonimmobilized protein. The N-terminal sequence of the immobilized protein on the PVDF membrane was determined. For proteins with a molecular mass less than 30 kDa, an overall yield between 10%-30% has been obtained.
已开发出一种蛋白质转移方法，该方法可将多肽洗脱并固定在聚偏二氟乙烯（PVDF）膜上。凝胶中的蛋白条带通过离心进行洗脱。离心印迹法包括以下步骤：（i）用1 M KCl在十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶中对蛋白质进行可视化，（ii）切下蛋白条带并在含有0.05% SDS/5%甲醇/0.02%二硫苏糖醇的蒸馏水溶液中平衡15分钟，（iii）将凝胶块与容器中的PVDF膜直接接触，（iv）在3000 g下离心1小时。在PVDF膜下方放置一个截留分子量为10 kDa的透析膜，以保留未固定的蛋白质。测定了PVDF膜上固定蛋白质的N端序列。对于分子量小于30 kDa的蛋白质，总产率在10%至30%之间。
