Band at same position using different primary antibodies - (Feb/03/2011 )
I ran 12% SDS PAGE using tris glycine buffer. after transfer i cut two lanes for ubiquitin detection and 4 lanes for beta actin. after development i got a strong intense single band for b actin. However for ubiquitin (8.5kDa) (polyclonal) i got one intense band and a few light bands. Problem is that the intense band is exactly at the same position as beta actin (46kDa). Now I am in doubt if the band was even specific for beta actin or was it some non specific band. What could be the reason for band at same position for two different proteins with very diferent mol weights.
Were you looking for Ub that is bound to something or were you looking for Ub by itself?
It may be that you are detecting Ub that is bound to one or more of the many proteins that are 40ish kDa.
bob1 on Thu Feb 3 23:28:20 2011 said:
Were you looking for Ub that is bound to something or were you looking for Ub by itself?
It may be that you are detecting Ub that is bound to one or more of the many proteins that are 40ish kDa.
well ubiquitin anitbody is polyclonal and i am looking for ubiquitin tagged proteins. so i was expecting multiple bands at all the molecular weight ranges. But the presence of extremely intense band a same position at same position as Actin has put me into doubt
It could be that the antibody has some non-specific activity or that it is being used at too high a concentration. Do you have a Ub tagged protein that you could titrate the antibody against to work out appropriate dilutions to use? Are you using proteasome inhibitors?