Casein Zymography- green/yellow line following dye front - (Sep/22/2006 )
I'm running Casein Zymography gels (12% Acrlyamide) on bovine extracts. When the dye front has run off the gel there is still a yellow/green line about 1/3- 1/2 way up the gel. We have ruled out temperature flucuations and gel composition as possible problems. It is most likely a pH problem and that is what I will be looking into next but I was wondering if anyone had experienced anything similiar and if so how was it rectified?
there should be some more information...what kind of gel-system/buffer-sytem is running? what is the dye? bromophenol blue?
I did run zymogram as well but never encounter those problem.
what's the sample buffer are you using?? I am using the sample buffer as applied in SDS-PAGE (containing bromophenol blue) but i never boil the samples. whats the voltage u used??
good result can be obtained at 100V..
The sample buffer that I am using is 150mM Tris (pH 6.8), 20% Glycerol, 0.02% bromophenol blue and 0.75% mercaptoethanol. The running buffer is 25mM Tris-HCl, 0.05% mercaptoethanol, 192mM glycine, 1mM EDTA and is pH to 8.3. I'm applying 100V for ~4 hours and running the entire system at 4 degrees C. The gels themselves are composed of 37:1 acrylamide:bisacrylamide, tris and water. The resolving gel contains 0.21% casein. I have not added SDS to any part of the system at this stage as SDS affects the activity of the calpains but I am going to try to add SDS to the sample buffer and running buffer this week and incubate the gel with Triton X prior to placing it in 10mM CaCl2 development buffer.
according to my casein zymography protocol, mercaptoethanol should be avoided. So, look for another protocol because I think you're wrong.
However, I'm doing a casein zymography but I have a problem. I can't get casein soluble. I read that casein can be soluble in a Tris solution (1,5 M ph=8.8) but my casein is not soluble in this tris solutuon.
Can you help me?
Thanks in advance