As usual, I run a 10 % SDS PAGE gel. However, problems appeared in my last two gels; the migration goes very slower than usual and the three upper bands of the EPM are well separate (which is unusual) and there is no bands below 50 kDa... So , no problem to see proteins above 50 but impossible to see any below 50 kDa...

I use the same parameters that always worked correctly. It could be that my gels are too concentrated but I did not change my concentrations...

Do you have any suggestions...

Thanks

Dear all,
I face a problem the last weeks I wanted to discuss with u.I extract total protein from cultured cells and I determine the concentration of the lysates by Bradford.It looks that my samples are protein-rich. The problem is that after carrying out the western blot, ponceau as well actin show that the protein amount I supposed to load is much lower.

In few words Bradford shows much protein and western only few. However I use other control proteins and these show that western blot and transfer work well.

I checked the reagents of my lysis buffer and the concentrations are compatible with bradford. So I was wondering what it may go wrong and interferes with bradford and how I could solve the problem.Any idea?

Thanks in advance for any help.

I am wondering if there is something to help use antibody raised in mouse on mouse tissues in Western Blot analysis?

I have to use a particular antibody and I am getting a lot of background and I think it's probably because of the same species.

For immunohisto there are several products that can help you use antibodies raised in mice on mouse tissues.

Is there anything like that for Western Blot analysis? There is only the one good antibody for my protein and therefore I am stuck using an antibody raised in mice on mouse tissues.

Special blocking agents or anything?

I am going to try to keep reducing the antibody concentration but there is only so low I can go.

I am trying to do a western blot analysis of the diaphragm tissue with alpha one a. I have received signal with B4 with the same tissue. I am getting no signal with alpha 1a. Does anyone have experience with this. The protocol is the same throughout the western blot except for different secondary antibodies.

Thanks
Amber