I am a chemical engineer and am told to purify a protein from a protein mixture. AND I know a little information about proteins. The isoelectric point of the desired protein is 8.91. I know if the buffer pH is above protein's isoelectric point, protein is charged negative. If it is below the isoelectric point, protein will be positively charged.
* If I use a buffer with pH=10 and a weak anion exchanger (DEAE-I have only anion exchanger) and then, decreasing the buffer pH gradually to slightly below the isoelectric point, can i take the desired protein? OR if use a cation exchanger, the buffer pH=8 is enough?
*** What should the content of the buffer be? for example, 5mM Na2HPO4, 1mM EDTA, 100mM NaCl and to obtain a pH=10, i will add NaOH to buffer solution???
Before the ion exchange chromatography, this procedure had been done by others and I think there are some unconscious addings of chemicals. I do not know the terminology of this type of science. excuse me. :-(
1. The desired protein was produced by bacteriums. Then, those bacteriums were put to -80 C.
2. Bacteriums were exploded with osmosis and then, centrifuged. supernatant was taken.
3. Urea, tris HCI, NaCl, triton solution (pH=8) was added to the supernatant and sonicated, then, centrifuged. this was done twice.
4. Guanidin HCl, NaCl, Mercaptoethanol, tris HCl solution (pH=8) was added to the supernatant and centrifuged.
5. DTT (Dithioeritrotriol) was added to the supernatant.
6. The protein was refolded by Na2HPO4, EDTA, Glutatyon solution (pH=8).
* It is known that increase in salt gradient releases the bounded proteins from ion exchange resins. why NaCl is used? Should it be dialysed? what should the dialysis buffer content be?
** What is the aim of these chemicals used in these steps?
All your advices are very crucial for me. Thank you very much. I am waiting for your help.
Edited by xaxax, 02 November 2011 - 01:55 AM.