EFFECTS OF THE MODIFICATION OF TRANSFER BUFFER COMPOSITION AND THE RENATURATION OF PROTEINS IN GELS ON THE RECOGNITION OF PROTEINS ON WESTERN BLOTS BY MONOCLONAL-ANTIBODIES
EFFECTS OF THE MODIFICATION OF TRANSFER BUFFER COMPOSITION AND THE RENATURATION OF PROTEINS IN GELS ON THE RECOGNITION OF PROTEINS ON WESTERN BLOTS BY MONOCLONAL-ANTIBODIES
复制标题
DOI：
10.1016/0003-2697(86)90207-1
复制
发表时间：
1986-08-15
影响因子：
2.9
通讯作者：
DUNN, SD
中科院分区：
文献类型：
--
作者：
DUNN, SD
已经开发了两种改进的蛋白质印迹法，以增强免疫化学识别。第一种是在pH 9.9的碳酸盐缓冲液中转移，而不是更常用的pH 8.3的Tris-甘氨酸缓冲液。这种改变提高了单克隆抗体对大肠杆菌F1-ATP酶五个亚基中四个亚基的识别，较小的亚基表现出最大的效果。碳酸盐缓冲液仅对最小的ATP酶亚基ε改善了多克隆抗体对连接到亚基上的二硝基苯基的识别。第二个修改是在温和的缓冲液中孵育凝胶，旨在促进蛋白质的复性，然后进行电泳转移步骤。最有效的缓冲液是在50 mM Tris-HCl中的20%甘油，pH 7.4。通过这种方法获得了用单克隆抗体获得的ATP酶所有亚基的信号的改善。由于亚基在大小、等电点和其他性质上有明显的差异，这种方法对大多数蛋白质都是有用的。15，000-Da ε的命运用125 I标记的亚基进行印迹实验。只要不向转移缓冲液中加入十二烷基硫酸钠，在Tris-甘氨酸或碳酸盐缓冲液中有效地与硝化纤维素结合。然而，ε.如果转移在碳酸盐缓冲液中进行，则在随后的孵育步骤中保留得更强。ε的结合当凝胶在转移前用缓冲甘油溶液处理时，对硝化纤维素的稳定性甚至更高。这些结果表明，ε.亚基第一次遇到硝化纤维显著影响随后步骤中结合的稳定性。通过用蛋白酶处理将F1-ATP酶部分片段化，然后在凝胶上电泳，或者立即转移到Tris-甘氨酸缓冲液中，或者用缓冲的甘油溶液处理并转移到碳酸盐缓冲液中。第二个印迹给出了对残余α的更强识别。亚基和片段被抗-α.单克隆抗体，对于较小的片段具有最大的改进。这一结果表明，修改后的程序可能是特别有用的，在提高小蛋白的检测。
Two modifications to Western blots which enhance immunochemical recognition have been developed. The first is transfer in carbonate buffer at pH 9.9, rather than the more commonly used Tris-glycine buffer at pH 8.3. This alteration improved the recognition of four of the five subunits of Escherichia coli F1-ATPase by monoclonal antibodies, the smaller subunits showing the greatest effects. Recognition of dinitrophenyl groups attached to the subunits by polyclonal antibodies was improved by the carbonate buffer only for the smallest ATPase subunit, .epsilon.. The second modification was incubation of the gel in mild buffers, designed to promote the renaturation of proteins, before the elelectrophoretic transfer step. The most effective buffer was 20% glycerol in 50 mM Tris-HCl, pH 7.4. Improvements in the signal obtained with monoclonal antibodies to all the subunits of ATPase were obtained by this procedure. As the subunits vary markedly in size, isoelectric point, and other properties, this method should be useful for most proteins. The fate of the 15,000-Da .epsilon. subunit, labeled with 125I, was followed through a blotting experiment. As long as no sodium dodecyl sulfate was added to the transfer buffer, .epsilon. was bound to nitrocellulose efficiently in either Tris-glycine or carbonate buffer. However, the .epsilon. was retained much more strongly during the subsequent incubation steps if the transfer was done in the carbonate buffer. The binding of .epsilon. to the nitrocellulose was even more stable when the gel had been treated with the buffered glycerol solution before transfer. These results indicate that the conditions under which .epsilon. subunit first encounters the nitrocellulose markedly affect the stability of binding during subsequent steps. The F1-ATPase was partially fragmented by treatment with proteases and then run on a gel and either transferred immediately in Tris-glycine buffer or else treated with the buffered glycerol solution and transferred in the carbonate buffer. The second blot gave stronger recognition of residual .alpha. subunit and fragments by an anti-.alpha. monoclonal antibody, with the largest improvement for the smaller fragments. This result suggests that the modified procedure may be particularly useful in enhancing the detection of small proteins.