I did a lot of Western Blot, and my band of lamin B1 always seem uneven.
I charge 30ug in 20uL by well, my samples are in different conditions but lamin B1 its the control. I don´t know what can I do.
The first four are from LNCaP cell and the second four are from VCaP cells.
It could be a difference between lamin B1 of these cell lines?
Hola Marian,
The first four bands in the first blot do look like "real" downregulation. However in the second blot they don't. Altogether it seems like a somewhat weak stain in both blots. I think it would be easiest to try another housekeeping control first, like Actin, or Gapdh and see what you get. If these look weird as well you have to look for further explanations.
Hope that helps.
hello Marian Cruz may be the problem in your sample preparation and loading
Hi Marian, you can try to use other housekeeping genes in parallel, as suggested above, beta-actin, GAPDH, tublin, etc. If they are all even, then it means the conditions you used probably affect the expression of Lamin B1. To determine which levels are affected, you can check the mRNA levels of Lamin B1 to see if they are changed; if not, the conditions may affect the protein levels of Lamin B1, either via post-translational modifications or affecting the protein degradation. If that happens, you cannot use Lamin B1 as your loading control anymore.
Hope it's helpful for you! Good luck!
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