diffuse bands on western blots - (Aug/14/2006 )

Hi all,

I've been running western blots on the biorad mini gel systems. I have loaded 20ug of protien and optimised antibodies. I ran a gel with 30ug of the same samples, on the same system and probed with the same antibodies O/N but I got diffused bands. I also have to expose the film for longer. I checked with ponceau and the transfer was good. The actin was fine.

If I increase the amount of loaded protien do I have to readjust antibody concentrations? Are the diffused bands due to too much protien loaded? is it generally better to load more protien?

Thanks for your help!

no, you don't have to change the amount of antibody.

-how long did you let the gel polymerize?

-an other point : detergent (triton X-100, NP40...) disturb the migration. By increasing the amount of protein, you increase the amount of detergent.
what was the amount of detergent you loaded?

I let the gel polymerise for about 10 minutes. I used 0.4% sds.

10 minutes is really too too short.
People say to let it 30 minutes. I'm a perfectionnist, and want thin bands. I never go under 3 hours.

I think this is the explaination.

Missele is right, you should at least polymerize the gel for 30 mins. If you want to do it faster, pour your gels the day before and put them in a plastic bag, covered with wet papers, than you have your gels ready and can start directly. Or you increase the amount of TEMED, then the polymerization is faster.

If you want to load more protein, you could try to run your gel in a cold room or with lower voltage for longer times, maybe it improves. But if it is working with 20 µg, why use more? I always use 25 µg and it works fine.

Molecular Cloning by Sambrook and Maniatis said leave it for 30 min.