Dear Ashwini,

Try to reduce the amount of protein you run on the gel by diluting your samples. Also reducing the running voltage will allow a smoother run of your sample.

Best,

Alexandros

Hello Ashwini,

I agree with Alexandros, about avoiding to run high amounts of protein at high voltage. Also what % gel are you using? Are you using purified protein samples or cellular extracts?

Best 

Aparajita

Ashiwini I think You can further reduce the contamination of other proteins by using membranes filters... First use of 10KD cutoff membrane which would bring your protein the permeate and all the proteins above 10KD would be left in retentate. Then pass your permeate through 3KD centrifugal filter so that your protein of interest would concentrate and protein less than 3 KD would be removed.... This would make your protein solution clearer (less contaminant proteins) and then you can run Tricine SDS PAGE.

Hello Aparajita,

The protein sample is purified fraction from a GPC G-25 superfine column. I am using 16.5% gel.

Thanks.

Hi Ashwini,

As Deepti suggested you can remove larger size proteins through a cut-off filter. In other case maybe use a low % gel.

If abovementioned suggestion does not help, you can try to run your samples in different electrophoresis system: Laemmli. If you use home made sample buffer, prepare the new one.