| 查看: 1673 | 回复: 2 | |||
yyc12596新虫 (著名写手)
|
[交流]
免疫共沉淀总是出现非特异性条带
|
|
目的蛋白直接用RIPA裂解液裂解（1%NP40），目的蛋白含Biotin. Streptavidin-beads做IP. WB用的目的蛋白抗体做一抗。 显影后，阴性组和阳性组出现类似的条带，应该都是非特异性条带。目的条带看不到。 |
回复此楼» 猜你喜欢
一志愿沪9，生物学326求调剂
已经有3人回复
-
药学求调剂
已经有10人回复
-
307中医考研调剂
已经有3人回复
-
271求调剂
已经有31人回复
-
291求调剂
已经有4人回复
-
山东省基金2026
已经有11人回复
-
一志愿华中农业071010，320求调剂
已经有10人回复
-
材料工程281还有调剂机会吗
已经有43人回复
-
291求调剂
已经有5人回复
-
085801电气专硕272求调剂
已经有18人回复
» 本主题相关价值贴推荐，对您同样有帮助:
- 求助Western Blot实验条带没出现的原因分析及解决办法
已经有7人回复
- 拟南芥蛋白互作非特异条带怎么解决？
已经有7人回复
- CO-IP还是pull down?
已经有5人回复
- Western Blot 目标条带跑不出来，内参条带清晰
已经有13人回复
- 免疫沉淀后进行western blot 应该有几条带？
已经有16人回复
- 【求助/交流】纯化酶切后的膜蛋白做Western blot没条带，why？
已经有9人回复
- 【求助/交流】请教Western blot出现非特异性条带的怎么解决
已经有8人回复
- 【求助/交流】pull down实验阴性对照也有带？
已经有5人回复
yyc12596
新虫 (著名写手)
- 应助: 10 (幼儿园)
- 金币: 10847.3
- 散金: 2230
- 红花: 34
- 帖子: 2270
- 在线: 355.1小时
- 虫号: 2034994
- 注册: 2012-09-28
- 性别: MM
- 专业: 白血病
2楼2015-02-24 03:03:12
yyc12596
新虫 (著名写手)
- 应助: 10 (幼儿园)
- 金币: 10847.3
- 散金: 2230
- 红花: 34
- 帖子: 2270
- 在线: 355.1小时
- 虫号: 2034994
- 注册: 2012-09-28
- 性别: MM
- 专业: 白血病
|
网上搜到的一些建议： You may want to start with preclearing your lysates. You incubate your lysates with rabbit IgG (bound to beads) to clear away any binding that nonspecifically occurs with the species antibody. I don't recommend the use of RIPA though. I think it's just too aggressive of a lysis buffer. I use a simple NP40 (1%) buffer to lyse cells for IP. I also prefer to bind the antibody to the bead, wash well and block in 5% BSA before adding the lysate (rather than adding the antibody to the lysate followed by beads). This helps get rid of nonspecific binding to the bead itself. Your washing is definately an issue as well. PBS just isn't going to do the job. Minimally you wash in the buffer you lysed the cells in. You can make more aggressive washes but 1M salt is WAY to high (Urea is very aggressive and will destroy everything). You will disrupt all the interactions. I start by taking the lysis buffer to 250mM and will go up to about 500mM. Additionally you can add a little more detergent but I suggest you do this in small steps to determine the best conditions. It's frustrating to run the gel and see that you washed everything away! The other idea is to use a wash buffer without the salt. Nonspecific interactions can often be through salt bridges. One quick wash in a no salt (or very low) will get rid of this background as well. You can try the washes alone or in combination as well. I had one IP that required a high detergent (high salt did nothing) followed by a no salt wash in order to remove nonspecific bands. You are just going to have to play around with the conditions and see what works best for this situation. I can recommend a book: Using Antibodies by Harlow and Lane. There are many good hint and tips for troubleshooting and this book covers practically all assays involving antibodies. |
3楼2015-02-26 01:46:07
