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No bands/weird bands on western blot - (Mar/11/2013 )

Hi, so this problem seems to have been going on for the last few weeks now.

Basically I have been doing westerns and getting no bands at all, I am staining for P-ATF2, but am also having trouble detecting the control B-actin. I have tried replacing the running buffer, transfer buffer, acrylamide stock, ECL, but to no avail. Other people have used those stocks and got them to work fine. I thought it might be my secondary antibody, but when using someone elses secondary, it still doesn't work.

I have used Ponceau staining and I can see plenty of lovely separated bands on my membrane.

My suspicion at the moment is that my box of antibodies may have been left out of the freezer for an extended period of time, but that wouldn't explain why the actin antibody I have borrowed from someone else didn't work properly either.

The attached image shows my latest attempt at getting something to stain, the single band you can see is supposed to be the Actin, after a 20 min exposure, but there is no reason why it is only appearing in one of the wells when all of the wells show good bands on the Ponceau stain!

I have another antibody to P-ATF2 that I borrowed from someone else, that I tried to re-stain the same membrane with after my one didn't work, but I didn't get anything with that either. (If I try staining with one antibody and it doesn't work, then try re-staining with a different antibody to the same thing, do I have to strip the membrane first to remove the original antibody?)

I really am running out of ideas now as to what could have gone wrong. I am certain the gel and blotting stages went well due to the good Ponceau staining, but have no idea where it could be going wrong after this. I have done lots of westerns before and never had this problem!
Attached Image


A couple of questions. Other people in the lab are seeing correct banding for the secondary Ab that you have found does not work? In a different set of hands, does the primary Ab work? All the buffers, post-transfer, are ubiquitously used throughout the lab? No problems are seen with these buffers?

In your picture, the top band is supposed to be B-actin? Is it supposed to extend across the whole blot? If it is supposed to run across the gel, have your ran coomassie stained to look at your loading concentrations?

It is weird because B-actin is usually so beautiful, no matter what companies Ab you use.


yeah the band in the image is supposed to be B actin, and it is supposed to be in all 7 wells not just one of them! The only other antibodies on the membrane were for P-ATF2 which is around 70kD, sorry I didn't mark the band sizes! I didn't use coomassie, but I use Ponceau staining quite alot, as it is reversible and with Ponceau I see lots of bands, of relitavely equal staining.

The rest of my lab hasn't wanted to try my secondary now that I am having problems! But someone is trying it out this afternoon with their loading control so we'll see if it works then.

No one else is looking for the ATF2 as I am so they haven't been using my antibody, but I could ask one of them to give it a go at some point. And the buffers aren't ubiquitously used, but we all use the same stock chemicals and recipes to make up our own buffers.


So weird