Wester blot non specific bands caused by secondary - (Apr/01/2008 )

Hello everyone, I'm doing a western blot on connexin 43 using Millipore anti-cx43 as the primary at 1:1000 and using Santa Cruz donkey-anti-mouse as the secondary. Basically I've been getting multiple non-specific bands and I've probed the blot alone with the secondary and it seems the secondary antibody is at fault. I've reduced the secondary antibody down to 1:1000000 and there is STILL some non-specific bands though they are quiet light. WTF? Does anyone have any suggestions? Any help will be great

---

If you think that the secondary ab is the problem, why not to try one from another company?? Maybe from the lab next to yours, you can borrow some??

We use anti-mouse IgG HRP-linked from Amersham NA931.

---

Thanks Zek, yeah that's my next port of call. I have some old Jackson Immuno anti-mouse. I haven't used it because there's only a few aliquots left. But it definitely works. So i'll just have to give it a go and cross my fingers!

---

Try to incubate in the presence of non fat milk or BSA. try several concentrations from 0.1% up to 3%

---