Electrophoresis Problems

[missmel]

Hello everybody..I have elecrophorised some whole cell extracts but I only got single huge bands in each lane, while the marker has run perfectly..Could anyone help me out to solve the problem?

Thank you..

[moleculardude]

Hey Missmel!

Welcome to the forums!

Wow thats a weird one. I think you have a problem with your sample preparation. It may be your SDS-PAGE loading buffer that is the problem.

If your sample buffer has too much sucrose this may cause the problem.

I would remake your SDS-Sample buffer or get some off someone that does not have this problem.

Also could be your samples themselves. Maybe too much protein or salt concentration.

Please give us a bit more information and we can help you more!
please tell us the outcome you got,
Cheers

[missmel]

Thanx for the quick response.
Now that you mention it, I use a non-reducing sample buffer (no mercaptoethanol, or DTT) 'cause the antibody detects a non-reduced epitope...
Could that be causing the problem? If yes, any suggestions as to how to bypass it?

thanx again

[mara1412]

Yes, this is your problem....
you should just reduce acrylamide % and use a non reduced marker....but you must remember that when you run non denatured samples, the migration is not only dependent from molecular weight, but also from secondary and tertiary structure of the protein.