LOSS OF ANTIGENS FROM IMMUNOBLOTTING MEMBRANES
LOSS OF ANTIGENS FROM IMMUNOBLOTTING MEMBRANES
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DOI：
10.1016/0022-1759(89)90343-8
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发表时间：
1989-08-15
影响因子：
2.2
通讯作者：
BEFUS, D
中科院分区：
文献类型：
--
作者：
DENHOLLANDER, N;BEFUS, D
page分离蛋白的免疫印迹法被广泛应用。从PAGE凝胶电泳转移蛋白质后，膜上未被占用的位点被过量的蛋白质阻断或淬灭。免疫印迹技术的灵敏度部分取决于与膜相关的抗原的量。我们已经研究了在用牛奶阻断各种膜时，印迹蛋白的损失。转印迹蛋白从硝化纤维素膜（NC）中丢失（0.45 .mu）。M)与堵塞溶液中牛奶的量成正比，损失随时间增加。当用5%的牛奶作为阻断溶液时，在1小时内损失的量约为10%，在24小时内接近25%。这种损失是非选择性的，涉及广泛的Mr.抗原。用四种免疫印迹膜：Immobilon-P （Millipore）、NitroPlus-2000 （MSI）和NC （0.2 .mu）检测了转印迹蛋白损失的时间和浓度依赖关系。M和0.45 .mu。M孔径)。NC (0.2 .mu。M)的效果最好，1 h时损失率最低（< 5%）。我们建议使用NC （0.2 .mu）。M)并用0.5%牛奶阻断30分钟，以尽量减少免疫印迹膜上抗原的损失。
Immunoblotting of PAGE-separated proteins is widely used. After electrophoretically transferring proteins from PAGE gels, sites on the membrane which are unoccupied are blocked or quenched with excess protein. The sensitivity of the immunoblotting technique is in part dependent on the amount of antigen associated with the membrane. We have investigated the loss of blotted proteins during blocking of various membranes with milk. Transblotted proteins are lost from nitrocellulose membranes (NC) (0.45 .mu.m) in prportion to the amount of milk in the blocking solution and loss increases with time. When 5% milk was used as the blocking solution the amount lost was about 10% within 1 h and approached 25% at 24 h. This loss was nonselective and involved blotted antigens of a wide range of Mr. This time- and concentration-dependent relationship for the loss of transblotted proteins was tested with four immunoblotting membranes: Immobilon-P (Millipore), NitroPlus-2000 (MSI), and NC (0.2 .mu.m and 0.45 .mu.m pore size). NC (0.2 .mu.m) gave the best results with the lowest percentage loss at 1 h (< 5%). We recommend using NC (0.2 .mu.m) and blocking with 0.5% milk for 30 min to minimize the loss of antigens from immunoblotting membranes.