Sudden problems with western-blots

[ScienceEnthusiast]

TL;DR

After changing secondary antibodies, western blots comes out badly

Hello, a problem from a newbie with western blots.


I've been doing western blots for a few months now, and everything was going alright. But 2 weeks ago, all blots started coming out badly as in the photos. Before, neither we had those black dots ont the sides of membranes, or those black blurs. They did started to show when we started using new batch of secondary antibodies tho. We've checked some things already, like different blocker, different washing regieme, blocking in PBST or in blocker, but none of them changed much. Also the exposition time during imagining changed from around 20-30 seconds to less than a second. Next week we will try different dillution of secondary antibodies, since with the old batch, we were using quite low dillutions (1:1000, they also were quite old), but before that i wanted to ask, if anyone maybe had similar problems.


Thank you for your time and interest.

 

[BillTre]

Do you filter or spin down your antibody solutions?
The new antibody maybe clumping of something. Ultra-filtration or centrifuging might remove clumps and leave the good stuff. I used to use filtration, but you lose some of the volume in the filter.

What does it look like if you leave out the new antibody, still a lot of background or clear?
There lots of controls you may have done already.

Do you pre-wash with BSA or serum from a species your secondary antibodies won't interact with?

 

[ScienceEnthusiast]

We havent' been spinning them, nor filtering, I think we will look into it. We haven't tried omitting the incubation with secondaries yet, will propably try it on monday. We block our membranes with 3% milk + 1% BSA solution, which shouldn't interact with our secondaries, if I understand properly what you're asking about.

And thanks for your interest and ideas.