Wrong imidazole conc

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    • #15606
      biology_06er
      Participant

      Hi there,

      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 using about 10mL of my 66mM conc and then basically collected any remaining bound protein with 10mL of 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!

      Thanks

      b_06er

    • #107464
      JackBean
      Participant
      quote biology_06er:

      Hope I’m making sense!

      not really

      but if your question is simply, whether you can pool fractions with your protein, than of course yes. Just take care so that you won’t pool all your franctions, then would be the chromatography quite useless. It depends, what is your aim. Is it lot of protein? Then look for protein activity. Is it pure protein (albeit in low amounts)? Then look for specific activity rather.

    • #107478
      canalon
      Participant

      Well from what I understand, 66mM of imidazole was enough to elute your protein so you do not need to add more, and you can use your elution to do all your experiments further. NAd next time you can use any concentration >66mM to elute your protein.
      And if I am right it appears that you were going through the motions of doing a gradient elution on you column (Ni-NTA I presume) but were not checking for the presence of the protein in each fraction to determine at which concentration the protein was leaving your column.

    • #107479
      JackBean
      Participant
      quote canalon:

      Well from what I understand, 66mM of imidazole was enough to elute your protein so you do not need to add more, and you can use your elution to do all your experiments further.

      I would agree, but he wrote, that the first time, he had the protein also in franctions 80 and 100 mM

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