Albiach M R, Guerri J, Moreno P
Instituto Valenciano de Investigaciones Agrarias (IVIA), Valencia, Spain.
Anal Biochem. 1994 Aug 15;221(1):25-8. doi: 10.1006/abio.1994.1373.
A simple procedure for reusing polyvinylidene difluoride membranes with bound proteins reacted with antibodies and developed by the nitro blue tetrazolium method (M. S. Blake, K. H. Johnston, G. J. Russell-Jones, and E. C. Gotschlich (1984) Anal. Biochem. 136, 175-179) is described. The blue diformazan precipitate produced by the reduction of the nitro blue tetrazolium salt was dissolved by soaking the membrane in dichloro- or trichloromethane for 10-60 min at room temperature. After equilibration in methanol and then in distilled water the first and second antibodies were removed by incubating the membrane in 0.2 M glycine-HCl buffer, pH 2.2, for 90 min at room temperature. These cleaned membranes could be successively reused with different monoclonal antibodies or preimmune serum up to 21 times. The intensity of the spots or electroblotted bands and their specific reaction pattern with different monoclonal antibodies remained unchanged, and no background was observed when the membranes were reacted with preimmune serum. This procedure makes it possible to reuse membranes freshly developed or stored for long periods without loss of resolution.
本文描述了一种简单的方法，可重复使用与抗体反应并通过硝基蓝四唑法显色的聚偏二氟乙烯膜（M. S. Blake、K. H. Johnston、G. J. Russell-Jones和E. C. Gotschlich，1984年，《分析生物化学》第136卷，第175 - 179页）。通过将膜在室温下于二氯甲烷或三氯甲烷中浸泡10 - 60分钟，可溶解由硝基蓝四唑盐还原产生的蓝色双甲臜沉淀。在甲醇中平衡后再在蒸馏水中平衡，然后通过在室温下将膜在pH 2.2的0.2 M甘氨酸 - HCl缓冲液中孵育90分钟来去除第一抗体和第二抗体。这些清洁后的膜可连续与不同的单克隆抗体或免疫前血清重复使用多达21次。斑点或电转印条带的强度以及它们与不同单克隆抗体的特异性反应模式保持不变，并且当膜与免疫前血清反应时未观察到背景。该方法使得可以重复使用新显色的或长期储存的膜而不会损失分辨率。
