4 replies to this topic

[ratnikasethi]

Hello Sir,
This is to request you to kindly guide me through a problem .I have a flag tagged protein and in order to purify it I have anti flag resin to follow immunoprecipitation . Now I need to elute my protein from resin in its native form with no harm on my protein. Other options available and provided are adding Flag peptide or 3X peptide for competitive inhibition is geting to costly. Also adding in enterokinase would not help..Kindly guide me through this hurdle as early as possible.as to how could I elute/seperate my Flag tagged protien after immunoprecipitation..
Thanking you

[Maxter]

Seems like you are doing affinity chromatography to purify the protein... You could try eluting with 0.2M Glycine-HCl pH 2.5. Glycine will provide with some buffering capabilities (since this pH is near its pK₁), and the low pH should disrupt the interaction between the Flag peptide and your antibody. The question now would be: "is your protein still folded at that pH". If it is an antibody (IgG) you should be fine. In any case, this is an option for a low cost elution. You could also elute this into a potassium buffer to immediately bring the protein into a more neutral pH. I usually use a small column and add 750uL glycine and elute into 250uL 1M K₂HPO₄. Collect 10-15 fractions, test with SDS-PAGE to see in which fractions you can find your protein.

Good luck.

[ratnikasethi]

Hello; thankyou so much for the reply..I am trying in for immunoprecipitation and not affinity chromatography..Now the question is that if protien seperates in this condition how would protien separate from the beads... ?

[Maxter]

the low pH should affect all protein-protein interaction

[rock&science]

Hi all, just wondering whether any of you guys have any experience using anti-FLAG M2 resin for column purification (instead of IP)?