Kurien Biji T, Scofield R Hal
Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK, 73104, USA,
Methods Mol Biol. 2015;1312:247-55. doi: 10.1007/978-1-4939-2694-7_26.
A method for the electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes following sodium dodecyl sulfate (SDS) polyacrylamide gel is described here. The transfer was performed with heated (70-75 °C) normal transfer buffer from which methanol had been omitted. Complete transfer of high and low molecular weight antigens (molecular weight protein standards, a purified protein, and proteins from a human tissue extract) could be carried out in 10 min for a 7 % (0.75 mm) SDS polyacrylamide gel. For 10 and 12.5 % gels (0.75 mm) the corresponding time was 15 min. A complete transfer could be carried out in 20 min for 7, 10, and 12.5 % gels (1.5 mm gels). The permeability of the gel is increased by heat, such that the proteins trapped in the polyacrylamide gel matrix can be easily transferred to the membrane. The heat mediated transfer method was compared with a conventional transfer protocol, under similar conditions. The conventional method transferred minimal low molecular weight proteins while retaining most of the high molecular weight proteins in the gel. In summary, this procedure is particularly useful for the transfer of high molecular weight proteins, very rapid, and avoids the use of methanol.
本文介绍了一种在十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶电泳后，将高分子量和低分子量蛋白质电转移至硝酸纤维素膜的方法。转移过程使用加热至70-75°C且省略了甲醇的常规转移缓冲液进行。对于7%（0.75mm）的SDS聚丙烯酰胺凝胶，高分子量和低分子量抗原（分子量蛋白标准品、一种纯化蛋白以及来自人体组织提取物的蛋白质）可在10分钟内完全转移。对于10%和12.5%的凝胶（0.75mm），相应时间为15分钟。对于7%、10%和12.5%的凝胶（1.5mm凝胶），20分钟内可实现完全转移。加热可增加凝胶的通透性，使被困在聚丙烯酰胺凝胶基质中的蛋白质能够轻松转移至膜上。在相似条件下，将热介导转移方法与传统转移方案进行了比较。传统方法转移的低分子量蛋白质极少，而大部分高分子量蛋白质仍保留在凝胶中。总之，该方法对于高分子量蛋白质的转移特别有用，速度非常快，且避免了使用甲醇。
