Cell Envelope Proteins Smeared - (Feb/22/2012 )
I am attempted to do a western on cell envelope protein I have isolated from P. aeruginosa, however the lower proteins always come out smeared. I have ran gels that are 10%, 12% and 15%. I store my protein in -20 with PMSF, I run at 80V for 30 min then 120 for 2.5 hours. My ladder is always clear. I have remade all of my buffers and have changed my sonication time and amplitude. I am at a loss.
Any input would be appreciated.
the smearing could be due to post-translational modifications on the protein (eg glycosylation, lipidation).
it could also be caused by components in the sample's buffer.