Wrong imidazole conc. - (Oct/29/2011 )
Recently I've been purifying a protein and the first time I did so, I used increasing concs of Imidazole--the lowest I used was 5mM and the highest 100mM and at both and also the concs. inbetween (10,20,40,80mM) protein was eluted. So the next time I purified the same protein I planned on eluting straight away with 100mM....I stupidly however, added 1mL of my 1000mM imidazole stock to 14mL of my buffer making it 66.6mM instead of 100mM (I should have had a final volume of 10mL)...I came to realise this as I was collecting my eluted protein...so I collected and then basically collected any remaining bound protein with 100mM...this is ok right?...I ran a gel and saw protein on the 66mM run but not on the 100mM (which is suprising so i'll run another one tomorrow)...but this is ok right? Even using the 66mM conc first and 100mM after is ok...last time I concentrated down my protein (after collecting 10mL from each conc=60mL) to about 10mL and used that for further purification using size exclusion. I can just do the same now right..say protein does show up on the 100mM run, I can combine the 66+100mM samples and concentrate protein right?
Hope I'm making sense!
it's okay (in fact, it's pretty routine, when running affinity chromatography, to ensure that elution is complete).
you may have nearly completely eluted the protein with the 66.6mM solution.