Microsequence analysis of peptides and proteins. VIII. Improved electroblotting of proteins onto membranes and derivatized glass-fiber sheets.
Microsequence analysis of peptides and proteins. VIII. Improved electroblotting of proteins onto membranes and derivatized glass-fiber sheets.
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肽和蛋白质的微序列分析。
DOI：
10.1016/0003-2697(88)90084-x
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发表时间：
1988
影响因子：
2.9
通讯作者：
Shively,JE
中科院分区：
文献类型：
--
作者：
Xu,QY;Shively,JE
我们定量研究了影响从十二烷基硫酸钠（SDS）凝胶到衍生化玻璃纤维纸或聚二氟乙烯（PVDF）膜的蛋白质的电转移和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% SDS凝胶转移到用QAPS （N-三甲氧基硅丙基-N，N，N-三甲基氯化铵）或APS（氨丙基三乙基氧基硅烷）衍生的玻璃纤维纸上，转移率可达90-95%。为了达到这些产量，有必要修改R. Aebersold等人（J. Biol）所描述的条件。化学，261,4229-4238,1986)。我们用稀氨水对玻璃纤维纸进行活化，用OAPS和APS在无水溶剂中进行衍生化，使其在衍生化过程中缓慢吸收水分。对于给定百分比的凝胶，转移率随转移时间和蛋白质分子量的变化而变化。在8%的凝胶上，高分子量的蛋白质获得了更短的转移时间和更高的收率。在相同的转移条件下，12%凝胶中分子量较低的蛋白质产率较高。转移蛋白的测序产率在10-80%之间，但在苯基硫代海因氨基酸衍生物的HPLC分析中观察到许多背景峰。在PVDF膜的类似实验中，转移率在85-95%之间。为了达到这些产量，有必要修改P. Matsudaira （J. Biol）所描述的条件。化学学报，2002,26(2):10035 - 10038,1987)。较低的电压和较长的转移时间可以获得较高的转移收率。为了获得持续的高转移收率，还需要在PVDF膜上预涂聚苯乙烯。PVDF膜被切割成大约1毫米宽的条，并插入到连续流动反应器中（J. E. Shively， P. Miller, and M. Ronk, Anal）。生物化学。163,517-525,1987)进行序列分析。样品装载到凝胶上，电转移到polybrencoated PVDF膜上，测序的总收率从β-乳球蛋白（10-50 pmol装载到SDS凝胶上）的50 - 60%到牛血清白蛋白和大豆胰蛋白酶抑制剂（50 pmol装载到SDS凝胶上）的20-30%不等。两种方法的比较表明，PVDF膜明显优于衍生化玻璃纤维纸，包括直接对考马塞蓝染色PVDF膜进行测序的能力，以及后续序列分析中观察到的较低背景。
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 OAPS 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 10–80%, 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 Polybrenecoated PVDF membranes, and sequenced ranged from 50–60% for β-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 the ability to directly sequence the Coomassie blue-stained PVDF membranes, and the lower backgrounds observed on subsequent sequence analysis.