Just a smear of proteins after ponceau red - Western Blot (Feb/20/2008 )
I loaded about 50-100µg cell lysate on a 6% SDS gel. I try to identify a 400kDa and low expressed protein by western blotting. The transfer using semi dy blotting seems to work (transfer buffer consisting of glycin, tris and methanol) and takes 105min at 0.4A. Afterwards I stain the membrane with Ponceau red straight away. The intensity is very good, but I cannot identify any bands rather a smear of proteins (the wohle lane).
My question is: Does it mean that the proteins are degraded or is that just normal?
Thanks for any help!
I think you loaded too much proteins.
50 µg is the maximum.
If the expression of your protein is too low, you should try to immunoprecipitate it.
I agree with missele, just try to use less protein or use a bigger gel size if you need to load so much of it. For western blotting, even for low expressed proteins, normally you don't need this vast amounts of protein if your antibody works well. If you need to establish the blot, try to use a positive control in a good gel system and then adjust your samples to it.
you may also want to try a gradient gel (eg 5-15%). this should help sharpen the bands. you may be seeing a lot of diffusion in the 6% gel (besides overload).
I agree with that. 6% is too low of a acrylamide concentration to get sharp bands as proteins can start to freely diffuse making this blurry. I use 7.5 % gels for large proteins and my lower molecular weight proteins are always a blurry mess but my high molecular weight proteins look fine. Why don't you try 10% or even 12% if you can't get your hands on a gradient gel for smaller molecular weight proteins. I think Novus has some precast gradient gel system samples if you want to try some.