I'm quite new at protein work, I hope these questions aren't too "stupid"
Actually i just started western blot on my his-tagged recombinant proteins. Using anti-his antibody on my cell lysate (from transformed bacteria) yields ~4 bands (the 1st lane from left). I guess i must be having truncated proteins. However, I then transfected my HEK 293T cells to produce the same his-tagged protein to see if it's truncated as well, the bands that appear seem to resemble an sds-page run! u can see in my picture the 3 right most lanes. i can't figure out what's the problem.
I block with 3-5% BSA in TBST and use anti-his Ab at 1:10,000 (it's already conjugated to AP so i don't need a secondary Ab). I treat my samples in 10%SDS/DTT.
Also, it's very hard to find skim milk powder in supermarkers here and the one we have now is stated 'high calcium'. I've been told that it will interfere with binding. May i know the reason why normal milk powder or full cream will not work? Will the fats interfere with blocking?
Thanks very much:D
Edited by littlebrownbat, 31 May 2011 - 12:57 AM.