By Jerome Champagne on Monday, June 16, 2003 - 03:16 am:
I am trying to purify proteins using sepharose-arginine. Apparently my target binds to the support but it seems I can not elute it with the classical methods (increase ionic strength, arginine). Is anybody experienced with this support?
By Anonymous on Monday, June 16, 2003 - 06:53 am:
Have you tried altering the pH of your mobile phase to elute instead?
By Jerome Champagne on Tuesday, June 17, 2003 - 12:27 am:
Yes, altering the pH doesn't work.
By HW Mueller on Tuesday, June 17, 2003 - 07:08 am:
No experience with that material, but proteins tend to bind to anything under various conditions. Have you tried chaotropes, detergents, organics, lowering ionic strength?
By Armando on Wednesday, June 25, 2003 - 04:58 am:
Jerome, I found these instructions in the amershambiosciences website:
Elution
Arginine Sepharose 4B is a group specific adsorbent
with affinity for a variety of biomolecules. Some
proteins interact biospecifically due to its structural
similarity with the ligand while others, bind in a less
specific manner by electrostatic interactions.
• Specifically bound biomolecules may be eluted
by competitive elution. Use of a competing
agent for either the ligand or the target
molecule, e.g. arginine in the buffer will elute
specifically bound substances.
• Less specifically bound biomolecules can be
eluted with increased ionic strength. Elution is
normally complete at salt concentrations of 2 M
or less of NaCl. Either step or continuous
gradients may be used.
• Reduction of the polarity of the elution buffer
by addition of dioxane (up to 10%) or ethylene
glycol (up to 50%) may be used for elution of
bound substances.
• An alternative for elution is to use urea or
guanidine hydrochloride which act as deforming
agents and elute bound substances.