Could someone suggest me any general purification method of Rtx toxin from liquid culture,As I am trying to purify one of the Rtx toxins through Hydrophobic Chromatography but I am getting a high background proteins.
I will first give a shot at imac using calcium or iron ions. Afterwards i will go for iex or hic.
You can also try ion-exchange chromatography on DEAE-Sepharose and phenyl-Sepharose
http://jb.asm.org/content/186/12/3760.full
Good luck
Dear Omar Gonzalez-Ortega and Aamal Ghazi Mahdi Al-Saadi thanks for your nice suggestions I have tried hic and it didn’t purify it at all, while the iex (using DEASE-Sepharose) worked for me but there is a very high background of other proteins (any idea how to avoid the background) and the protein is inactive even in presence of 2 M Ca+ . What could be the possible reasons that the toxin is inactive???
Valuable suggestions would be appreciated. Thanks.
IEX is only selective in charge, did you run a gradient for desorption? Did you try imac as I mentioned before? Was the toxin inactive before purification?
Dear Omar Gonzalez-Ortega I didn't run any desorption gradient and the toxin was active before purification, point regarding use of imac, I am not sure whether I can use it for native Rtx or not ? as it's W.T toxin expressed by the native bug itself (not a recombinant one, which could be tagged with any marker in imac purification)....
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