Hi Andrea,

Do you have input lysates run on a separate gel for your IP which did not work? How does that compare to the one that worked? Another question remains is that do you use any reagent to remove your antibodies after your IP before running the gels because I do not see any sign of heavy chain or light chain in your membranes.

Once you answer these questions I might be able to help you further.

Best,

Ayan

Hi there,

All the samples have been prepared at the same time using the same reagents? Are both gels from the same prep? The facts that some samples are not actually electrofocusing in the concentration gel suggests there is either something wrong with their composition (loading buffer) and/or gel composition. total proteolysis of these samples might also explain the situation...

It seems a problem related with the samples and not with the gel preparation, since molecular weight markers are well resolved. Have you used the same sample buffer for both gels, or did you perform any different treatment for the samples as temperature, or reduction agents, etc, before loading the gel?

Usually it's a sample problem. Such as degradation of protein, loading buffer, contamination of sample... Anyway, you should re-started from your cells and make sure everything you need is fine. Also it will be easier to find out the reason.

It sort of looks like you haven't gotten all the Ponceau washed off of the membrane. Is that possible ?

Why the MWs pattern are totally different?

Thank you for the answers!

Ayan Chanda I did only total separately, and you can see it as the last one on the membrane. It never worked when done with the rest of the problematic samples. But also here it is much weaker than the other samples, so I guess that the proteins are degraded during IP. After O/N incubation I wash beads 3x and elute in SDS buffer.

Dominique Liger Samples are from the same IP, done with the same reagents. I also thought that my gels are the problem, so I tested precast ones and it did not work, so the samples are bad definitely. María Luisa Caballero

Maureen Lynch I tried longer destaining, and I can destain it but later on when visualizing proteins I also have columns as you see on membrane..

I've also tested mock cells pellets on the gels I made, and it worked perfectly, so I assume that something is wrong with the samples but as my first IP was successful, and I did all the same, I don't know what can be the problem.

Attila Lehotzky Because I have different samples here

In my opinion, residues of organic solvents (ethanolor whatever) as well as compounds affecting a correct pH of the sample buffer may cause a non-uniform run of protein samples in your western blot.

I would propose to carefully revise your protocol and check all used solutions/buffers for correct composition and pH.