I got this SDS PAGE gel after running about 3 hours with tris tricine buffer system. Half of the gel showed the separation of protein, but the others showed the blank band. I'm not sure wether all of the band is end at the middle of the gel, or there are other reason. Could you give me an idea?
Do you mean that the bottom part of your gel is not containing separated proteins after your - I guess - coomassie staining? For me it looks like the loaded sample just didn't run until the end of the gel. What happened to your dye at the running front? Have you seen it running out of the gel? And another question: Are you working with a purified protein or with a full lysate?
It seems to be overrun, since the protein reach the other side of the wells. When the electrophoresis is overrun the protein will escape from the gel and go to the positive electrode. Perhaps you should reduce the running time by 0.5-1.5 hours. I think you could optimize the running time simply by watching the movement of the dye within the loading buffer when running.
you did not mention that what percentage of SDS-PAGE you used. In your image, it seems that gel is not run completely. In this case, I guess you have stop the run when dye front came out.
Tricine -SDS is specially for seperation of small peptides. you have to load pre-stained marker in one well and run this gel initially at 30V for 2hrs and then you have to increase the voltage upto 150 volt. If you are using 16% of SDS-PAGE it will take at least 5-6 hr to reach the small peptides ( 1kDa) at the bottom but the dye will go out soon. So don't care about the dye front check with prestained protein marker. So via using Pre stained marker you can stop the running of gel according to your protein of interest. I am attaching a publication for the details of Tricine-SDS PAGE.
- Badges
- Science method