Protein is dissolved in larger volume of buffer (ammonium acetate). Thank you.
Dear Janani, Please give some more details about your protein size, isoelectric point, ammonium acetate concentration. The total volume of your sample with protein concentration… These details are essential to help you without the risk my advice will destroy your work.
Sir, My protein size is 16, Kda, Isoelectric point 7.4. I have finished the steps of dialysis, chromatagrphy of both anionic and cationic. and checked my protein presence using SDS PAGE also. But my protein is dissolved in buffer, that is used during chromatography. I want to elute the protein out of my buffer. Thankyou Sir.
Lyophilization as Amrit suggested might be best as ammonium acetate is volatile, and would come off with the water under vacuum. So freeze it, and put it under vacuum.
That would be choice 1. Choice 2 is to do a spin column to exchange the buffer, if:
1. You want the same concentration of protein
2. You want a different buffer.
If your protein needs to be concentrated, then a ultra amicon filter spin column would be appropriate with a membrane mw cutoff of 5 KDa. Then I would do a P10 or G 25 spin column for buffer exchange.
It depends on what you want to concentrate the protein for. For example, if the next step was trypsinolysis/de novo sequencing, you are interested in capturing protein, but do not care about biological activity. If, on the other hand, you want active protein back, then you need to concentrate differently. There are lots of old tricks out there. e.g. put protein solution into a dialysis tube and role the tube in dry Sephadex (it removes the water and buffer as it hydrates - messy, but it works!) But, your protein is small, and may not be an ideal candidate for this trick. If you have a large volume, be aware of the potential of a high resolution ion exchange step to concentrate a protein.
You don't really provide enough context and background to receive anything but the most generic advice.
Thankyou very much,Paul Lesbats Sir, Amr Negm Sir, Jan Piwowarski Sir, Marcia Moss Sir, Rob Beynon Sir, for your valuable suggestion, and the tricks I can now employ and proceed the next step in my research.
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