Department of Sciences & Mathematics, Mississippi University for Women, Columbus, MS 39701, USA.
Molecular Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Biotechniques. 2020 Jun;68(6):318-324. doi: 10.2144/btn-2019-0124. Epub 2020 Apr 14.
Protein samples electroblotted onto nitrocellulose membranes and quenched with a mixture of blocking agents produced a strong signal for cystic fibrosis transmembrane-conductance regulator (CFTR), a high-molecular-weight protein, in western blotting. Optimized conditions for CFTR were then extended to medium- and low-molecular-weight proteins (LAMP1 and Rab11a, respectively) to determine the effects of methanol concentration (0-20%) in Towbin's transfer buffer (TTB). Methanol in TTB appears to have little to no effect on CFTR signal. However, for medium-sized (LAMP1) and small (Rab11a) proteins, a lower concentration of methanol (10%) was sufficient to produce a maximal signal. Therefore, methanol, a toxic solvent, can be removed from or reduced in TTB without compromising signal strength. Here, we show modifications that may be useful in detecting and/or improving the signal of low-abundance proteins.
蛋白质样品经电转移至硝酸纤维素膜，并用封闭剂混合物淬灭后，在 Western blot 中产生了囊性纤维化跨膜电导调节剂（CFTR）的强信号，CFTR 是一种高分子量蛋白。然后将 CFTR 的优化条件扩展到中分子量和低分子量蛋白（分别为 LAMP1 和 Rab11a），以确定 Towbin 转移缓冲液（TTB）中甲醇浓度（0-20%）的影响。TTB 中的甲醇似乎对 CFTR 信号几乎没有影响。然而，对于中等大小（LAMP1）和小（Rab11a）蛋白，甲醇浓度（10%）较低足以产生最大信号。因此，可以在不影响信号强度的情况下从 TTB 中去除或减少甲醇这种有毒溶剂。在这里，我们展示了可能有助于检测和/或提高低丰度蛋白信号的修饰。
