My Protein is Cadmium binding and around 25 KDa molecular weight. PI is 4. 76. Can anybody recommend me which column and chromatography should I use for protein purification. Its soluble protein. I have taken its expression in ammonium sulphate precipitation and Dialysis also. Kindly guide me about its purification.
HPLC method was used for determination of Metallothionein -I and Metallothionein-ll. MTs were separated by an HPLC exchange column, eluted through a linear gradient of buffer and the peak containing Metallothionein was determined by AAS.
Thank you so much Sullivan Lee
for your kind reply. Kindly let me know is it company that provide these services for Biological Samples? Is it Paid process?
FPLC systems are made from bio-compatible materials (glass, plastic), whilest HPLC systems are made from steel. Also, FPLC systems are preparative scale, HPLC more analytical (although there is overlap).
Given that your protein has a low pI, purification by anion exchange chromatography at a pH about one unit higher than the pI seems a logical starting point. Your protein will still have a net negative charge, most other proteins have a higher pI and hence will be positively charged and not bind to the column. Thus, you can use a small column for a large amount of feed, and elute the protein later in a very concentrated form.
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