【求助】用了PIERCE的免疫沉淀试剂盒，多次失败，想请大家帮忙找一下原因，谢谢

按照说明书，取了50ug抗体（多抗），固定于50ul的凝胶上，取1/5量与含有约500ug总蛋白的样本反映，洗脱液用western blot检测，未见条带。实验步骤严格按照说明书进行。

1. Does the Ab suitable for IP?2. Are you sure the Ab had been coupled on to the Gel?3. During wetern blot, can your positive control be seen?4. Run the cell lysat that after IP and do WB to check target protein binding to Ab-gel or not.

谢谢你的回复：1、我的抗体说明书上说是可以做免疫沉淀的2、用分光光度计对抗体结合前后进行光密度的测量： 分别是2.710A和0.041A，可见抗体应该已经结合到凝胶上了3、用WB检测时，存在阳性对照；但是在IP的洗脱液中，没有检测到任何蛋白条带

1. For IP, mAb is better. For multiple Ab, about 5% is the Target IgG. I think you can use double Ab and cell lysates to try again.2. Does your cell express the target protein in high lelvel? (If not. give some stimulation to increase the expression).3. The elution buffer in the kit is very soft since the Ab-Gel will be re-use. I also found the effect of elution is very low of this kit, if your target protein bind to Ab hargly. I surggest you try 0.1%SDS to elution (3 times: 1st: RT 15 min, 2nd 37 C 15 min, 3rd: 65 C 15 min). In this case, your beads can not be re-use. But you can check the elution effict.4. Staining your membrane by 丽春红 before blot.5. Run the cell lysat that befoer and after IP and do WB to check target protein binding to Ab-gel or not.6. After transfer protein to membrane, Stain the gel by silver staining method. you can see the transfer effect. Good luck!