I did affinity chromatography with GFP protein, Histidine tag and Imidazole buffer.
I did washing with 20mM Imidazole buffer to eliminate non-specific proteins but found out that it luminesces under UV lamp which means also GFP has been eluted. I don't know why🥲 Can someone explain me the reason..??
The one that I circled in the picture is the washing result!
I have purified hisTag-GFP by IMAC Nickel. The yield is so gross, for that reason you should use a 5 ml column if you want to catch all of them. Even when you would recover a lot GFP in the elution, it overload the column and you can see it in the wash fraction too.
imidazole is used to elute the strongest binding species in your mixture: the His tagged GFP, so it makes sense the so called "stringent wash" of 20mM imidazole might release some too, try washing to baseline (no A280nm signal or GFP florescence) with the loading buffer first , then wash again with lower imidazole concentrations (check for GFP in wash) and stepwise increase until you see contaminant proteins but not GFP; its a balancing act! Good luck!
Certainly, column overload should be the point you need to check first. You can note that the concentration of the flow-through fraction is less in comparison to eluted ones. This may indicate, that you probably exceed the dynamic binding range of the resin. Load the FT fraction again to the column and observe for any washed-out GFP, this verifies the concentration overload...
Emir
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