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elution sample form Mono Q column


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#1 Poteo

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Posted 22 February 2005 - 08:35 PM

Dear, everybody
I loaded my sample; halophilic proteinase contained high negative charge in the surface of molecules, in Mono Q column. I could not eluted the sample form column, eventhough 4M NaCl in 50 mM Tris-HCl, pH 8.0 + 1 mM CaCl2, were used. Please introduced me, what is the suitable buffer for eluted sample


Thank everybody for the good recommentation.

#2 sharath

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Posted 22 February 2005 - 08:58 PM

ph 8.0 is too high for ion exchange chromatography, especially in your case since it has negatively charge. try at 7.0 or even at 6.0 if the protein is stable at those pH.
Sharath B.

#3 speeder

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Posted 23 February 2005 - 03:21 AM

If you have made sure that your protein stayed on the column and did not come off in the flow through or the wash, then I would go to KCl which seems to work well at same concentration or a saturated solution. You can also try to decrease the pH of the buffer to decrease the negative charge on the surface of the protein as sharath stated.

#4 ZDF

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Posted 23 February 2005 - 12:42 PM

If you cannot elute your protein in 2M of salt you cannot do it at all. Try 7M GuHCl+1M NaCl. I think your protein unstable at low ionic strange and form aggregates at loading on a column. It is not unusual situation with halophilic proteins. Look Zaccai's papers about it.

Edited by ZDF, 23 February 2005 - 12:43 PM.


#5 sharath

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Posted 24 February 2005 - 08:07 PM

The role of salt in your protein appears to be complicated. Better option would be to try other methods like hydrophobic interaction chromatography, affinity or gel filtration.

Sharath.B
Natioanl Chemical Laboratory,
India
Sharath B.




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