Extra bands after protein expression/purification
Posted 09 March 2012 - 12:12 PM
I used 0.5 mM IPTG to induce a 1.0 OD culture (750ml)
Would it help to lower the IPTG and maybe do a 0.6 OD culture? I induce at 20C overnight
Any advise would be greatly appreciated!
Posted 10 March 2012 - 12:34 AM
Posted 12 March 2012 - 06:38 AM
lower molecular weight possibly means cleavage by proteases ...do you use protease inhibitor?
I do not use protease inhibitor because the protein of interest has protease activity and I do not want to disrupt that. Also, this protein cannot self-cleave since the sites needed are disrupted
Posted 12 March 2012 - 01:10 PM
If you still suffer from degradation you will have to purify your protein after His-Trapping by size exclusion to get rid of the degradation products.
Posted 12 March 2012 - 01:35 PM
I did run the sample through a millipore 30,000 MW filter and it appeared to not remove any contaminants! the 30,000 MW is well below my protein weight ~70,000 and should have removed most contaminating bands. After running on a gel, the filtrate and the fraction that was stopped by the filter look identical. Another student in my lab told me the filters were unreliable and I have to agree. Has anyone used these millipore microcon filters?