"Diffuesed" bands of higher molecular weight - (Jan/21/2010 )

Hi everybody,
I have a problem with my western blot. I try to detect 3 different proteins on the same membrane 40, 76 and 95 kDa. For this I run 7,5% gel. The problem is that the higher the molecular weight is the more ugly diffused bands I get. At 40 kDa they are quite OK, but at 96 they are terrible. Does anybody know what the problem could be. The tricky thing is that sometimes all the bands are perfect and I really cannot understand what I did wrong. The one thing I change from time to time is the power supply, I was sure that one is better but than I had a bad gel with it, so noW I don't know. I have repeated it alredy twice with the same samples and now I ordered precast gels, because I do not have so many samples left. But I would really like to solve this problem without always using precast gells. Thanks a lot!

are you sure that your gel was fully polymerized?

are you using a stacking gel? this helps to sharpen banding.

also...

how long was the top of your gel exposed to buffer before you ran it? the top of the gel may have swollen.

one way to ensure sharp bands throughout the gel is to run a gradient gel (5-15%, 4-20%, etc.).

mdfenko on Jan 21 2010, 06:41 PM said:

thanks for the reply.
I use stacking gel. I exposed the gel to the buffer for let's say 15 min (5 min before loading of the samples and 10 min during the loading). Should I reduce this time?
I have never used gradient gels, but I will definitely try it

i remove the comb, add the buffer, load the lanes and start the gel. total time is 5-10 minutes, depending on how many lanes (and how straight the lanes are after removing the comb).

I normally run 8% gels and I detect proteins from 35 to 200 kda without any problem. So maybe you load too much protein onto your gel or you run the gels too fast.

And about the exposicion with the buffer, I normally storage gels in the fridge up to 3-6 days in running buffer, and they work as well as "fresh" gels.

mdfenko did you have any problems with the exposicion time to the buffer?? what happens? the proteins dont run normally? I ask because I never had (or I never noticed) that before, and just in case...

laurequillo on Jan 29 2010, 12:59 PM said:

i never store the gels in buffer. if i store the gel, i wrap the gel with moist paper towels and plastic wrap and store at 4C (without stacking gel).

most of the time i just use them right away.