Xu Q Y, Shively J E
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Anal Biochem. 1988 Apr;170(1):19-30. doi: 10.1016/0003-2697(88)90084-x.
We have quantitatively examined the various parameters affecting the electrotransfer and sequence analysis of proteins from sodium dodecyl sulfate (SDS) gels to derivatized glass fiber paper or to polyvinyldifluoride (PVDF) membranes. Transfer yields in the range of 90-95% can be obtained for proteins in the molecular weight range of 10-90 kDa for transfer from 12% SDS gels to glass fiber paper derivatized with either QAPS (N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride) or APS (aminopropyltriethoxysilane). In order to achieve these yields, it was necessary to modify the conditions described by R. Aebersold et al. (J. Biol. Chem. 261, 4229-4238, 1986). We activated the glass fiber paper with dilute ammonia water and derivatized the activated glass fiber paper with QAPS and APS in anhydrous solvents which were allowed to slowly absorb moisture during the derivatization process. The transfer yield varied with transfer time versus molecular weight of the protein for a given percentage gel. Shorter transfer times and higher yields were obtained for higher molecular weight proteins on 8% gels. Lower molecular weight protein gave higher yields from 12% gels under similar transfer conditions. Sequencing yields of the transferred proteins were in the range of 40-80%, but a number of background peaks were observed on HPLC analysis of the phenylthiohydantoin amino acid derivatives. Transfer yields in the range of 85-95% were observed for similar experiments with PVDF membranes. In order to achieve these yields, it was necessary to modify the conditions described by P. Matsudaira (J. Biol. Chem. 262, 10035-10038, 1987). A lower voltage and longer transfer times gave higher transfer yields. In order to achieve consistently high transfer yields, it was also necessary to precoat the PVDF membranes with Polybrene. The PVDF membranes were cut into approximately 1-mm-wide strips and inserted into a continuous flow reactor (J. E. Shively, P. Miller, and M. Ronk, Anal. Biochem. 163, 517-525, 1987) for sequence analysis. Overall yields of samples loaded onto gels, electrotransferred to Polybrene-coated PVDF membranes, and sequenced ranged from 50-60% for beta-lactoglobin (10-50 pmol loaded onto SDS gels) to 20-30% for bovine serum albumin and soybean trypsin inhibitor (50 pmol loaded onto SDS gels). A comparison of the two methods shows clear advantages for the PVDF membranes over the derivatized glass fiber paper, including the ability to directly sequence the Coomassie blue-stained PVDF membranes, and the lower backgrounds observed on subsequent sequence analysis.
我们已经定量研究了影响蛋白质从十二烷基硫酸钠（SDS）凝胶转移至衍生化玻璃纤维纸或聚偏二氟乙烯（PVDF）膜上进行电转移及序列分析的各种参数。对于分子量在10 - 90 kDa范围内的蛋白质，从12%的SDS凝胶转移至用QAPS（N - 三甲氧基硅丙基 - N,N,N - 三甲基氯化铵）或APS（氨丙基三乙氧基硅烷）衍生化的玻璃纤维纸上，转移产率可达90 - 95%。为了达到这些产率，有必要对R. Aebersold等人（《生物化学杂志》261, 4229 - 4238, 1986）所描述的条件进行修改。我们用稀氨水活化玻璃纤维纸，并在无水溶剂中用QAPS和APS对活化后的玻璃纤维纸进行衍生化，在衍生化过程中让溶剂缓慢吸收水分。对于给定百分比的凝胶，转移产率随转移时间和蛋白质分子量而变化。在8%的凝胶上，分子量较高的蛋白质转移时间较短且产率较高。在类似的转移条件下，分子量较低的蛋白质从12%的凝胶转移时产率较高。转移后蛋白质的测序产率在40 - 80%范围内，但在用苯硫基乙内酰脲氨基酸衍生物进行HPLC分析时观察到一些背景峰。用PVDF膜进行类似实验时，转移产率在85 - 95%范围内。为了达到这些产率，有必要对P. Matsudaira（《生物化学杂志》262, 10035 - 10038, 1987）所描述的条件进行修改。较低的电压和较长的转移时间可获得较高的转移产率。为了始终获得高转移产率，还需要用聚凝胺预涂PVDF膜。将PVDF膜切成约1毫米宽的条带，插入连续流动反应器（J. E. Shively、P. Miller和M. Ronk，《分析生物化学》163, 517 - 525, 1987）中进行序列分析。加载到凝胶上、电转移至涂有聚凝胺的PVDF膜上并进行测序的样品总产率，对于β - 乳球蛋白（加载到SDS凝胶上10 - 50皮摩尔）为50 - 60%，对于牛血清白蛋白和大豆胰蛋白酶抑制剂（加载到SDS凝胶上50皮摩尔）为20 - 30%。两种方法的比较表明，PVDF膜相对于衍生化玻璃纤维纸具有明显优势，包括能够直接对考马斯亮蓝染色的PVDF膜进行测序，以及在后续序列分析中观察到较低的背景。
