小中大
正好今天我也在第一次做这个实验，步骤按着这个程序做的，有经验的人帮我看看。
1、 Add 3 ml ice cold RIPA buffer to cell monolayer and incubate at 4° C for
10 minutes. For suspension cells, add the RIPA buffer to washed cell pellet
in a 15 ml conical centrifuge tube.
2、Disrupt cells by repeated aspiration through a 21 gauge needle and transfer
to a 15 ml conical centrifuge tube.
3、Pellet cellular debris by centrifu gation at 10,000xg for 10 minutes at 4° C.
Transfer supernatant to a fresh 15 ml conical centrifuge tube on ice.
Preclear lysate (optional step) by adding 1.0 μg of the appropriate control
IgG (normal mouse, rat, rabbit or goat IgG, corresponding to the host
species of the primary antibody), together with 20 μl of resuspended
volume of Protein A/G PLUS-Agarose. Incubate at 4° C for 30 minutes.
Pellet beads by centrifu gation at 2,500 rpm (approximately 1,000xg) for 5
minutes at 4° C. Transfer supernatant (cell lysate) to a fresh 15 ml conical
centrifuge tube on ice.
4、Transfer 1 ml of the above cell lysate, or approximately 100–500 μg total
cellular protein, to a 1.5 ml microcentrifuge tube. Add 1–10 μl (i.e., 0.2–2
μg) primary antibody (optimal antibody concentration should be determined
by titration) and incubate for 1 hour at 4° C.
5、Add 20 μl of resuspended volume of Protein A/G PLUS-Agarose. Cap tubes
and incubate at 4° C on a rocker platform or rotating device for 1 hour to
overnight.
6、Collect immunoprecipitates by centrifugation at 2,500 rpm (approx imately
1,000xg) for 5 minutes at 4° C. Carefully aspirate and discard radioactive
supernatant.
7、 Wash pellet 4 times with 1.0 ml RIPA buffer (more stringent) or PBS (less
stringent), each time repeating centrifugation step above.
8、After final wash, aspirate and discard supernatant and resuspend pellet in
40 μl of 1x electrophoresis sample buffer.
9、Boil samples for 2–3 minutes and analyze 20 μl aliquots by SDS-PAGE and
autoradiography. Unused samples may be stored at -20° C.
10、 Optional: After boiling, samples may be centrifuged to pellet the agarose
beads followed by SDS-PAGE analysis of the supernatant.
不能理解第三步中
Preclear lysate (optional step) by adding 1.0 μg of the appropriate control
IgG (normal mouse, rat, rabbit or goat IgG, corresponding to the host
species of the primary antibody), together with 20 μl of resuspended
volume of Protein A/G PLUS-Agarose. Incubate at 4° C for 30 minutes.
Pellet beads by centrifu gation at 2,500 rpm (approximately 1,000xg) for 5
minutes at 4° C. Transfer supernatant (cell lysate) to a fresh 15 ml conical
centrifuge tube on ice.
这是什么作用？肯定不是对照了啰！！！
第五步，照上面战友的说法孵育的温度、时间、摇床的转速都是很重要的哦？我还准备就在室温呢，会增加非特异性结合？
抗体的量以及总蛋白的量（或者说他们的比值）重要不？需要严格控制不？还是需要调整？
买的是santa cruz的抗体，听说是没有验证过的，但说明书上写明了是可以进行免疫共沉淀的。
心里没底阿。
谢谢
