02-2012级硕士高级免疫学实验技术-Western.pptVIP

Example of immunoblotting 免疫学检测实验步骤 封闭：放入封闭液（含3%BSA的PBS-T） 10ml 中封闭 ，先在摇床上摇10min，然后放入37 ℃孵箱中放置60min。 孵育结束后，直接向封闭液中加入靶抗原特异的一抗（1:100），先在摇床上摇10min，再放入 37℃孵箱中孵育30min.　 用PBS-T洗三次，每次5min (在摇床上摇)。 弃去洗涤液，加入用1%BSA-PBS-T稀释的酶标二抗(HRP-DaR) 1:500，先在摇床上摇10min，再放入 37℃孵箱中孵育30min。 用PBS-T洗三次，每次5min (在摇床上摇)。 加入新配制的DAB底物液，显色，待显出明显条带时用dH2O终止反应。 The electrophoretic transfer of proteins from sodium dodecyl sulphate polyacrylamide gels (SDS) to sheets of nitrocellulose was initially referred to as “Western” blotting. In order to avoid this geographical jargon, the term immunoblotting is used. 不连续胶的系统使用(积层胶/分离胶)导致稀释的浓缩，并且能形成锐利的带形 丙烯酰胺凝胶用过硫酸铵聚合后，并且TEMED加速催化AP的自由基。因为氧能抑制胶的聚合过程，所以聚合过程除去气泡，加快聚合作用。 不连续胶的系统使用(积层胶/分离胶)导致稀释的浓缩，并且能形成锐利的带形 丙烯酰胺凝胶用过硫酸铵聚合后，并且TEMED加速催化AP的自由基。因为氧能抑制胶的聚合过程，所以聚合过程除去气泡，加快聚合作用。 * 1. Build the transfer “sandwich” onto the anode (-) plate as follows: ? 4 sheets blotting paper soaked in Transfer Buffer (10 min) Gel soaked in Transfer Buffer (10 min) NC membrane pre-wet with Methanol (1 min), then soaked in Transfer Buffer(10 min) 4 sheets blotting paper soaked in Transfer Buffer (10 min) 2. Carry out the transfer at 0.8mA per cm2 of gel area for about 2 hours at room temperature. * 1. Build the transfer “sandwich” onto the anode (-) plate as follows: ? 4 sheets blotting paper soaked in Transfer Buffer (10 min) Gel soaked in Transfer Buffer (10 min) NC membrane pre-wet with Methanol (1 min), then soaked in Transfer Buffer(10 min) 4 sheets blotting paper soaked in Transfer Buffer (10 min) 2. Carry out the transfer at 0.8mA per cm2 of gel area for about 2 hours at room temperature. * Immuno-Detection 1.??? Remove the NC membrane (blot) from the apparatus, block NC blot using 5% w/v BSA in PBS, overnight at 40C or 2 hours at room temperature. 2.??? Overlay the blot with 5 ml primary antibody at appropriate dilution (generally 1:50-1:500). Incubate for 2 hours at room temperature on shaker. 3.??? Wash the NC blot three times for 5 min with PBS-T 4.?? Incubate the blo