I'm purifying a recombinant protein. Currently, I use HIC to purify it. The specification of my Bind and Elution buffer is according to the following concentration, EC and pH...
Bind,
Sodium phosphate 30mM
EC 60
pH 7.5
Elation,
Sodium phosphate 5mM
EC 60
pH 8.5
When I washing the column with Elution buffer, impurities and som of my target proteins are removed. However, most of my target protein is removed during washing with water, where EC is reduced to less than 0.5 mS.
Here I'm looking for buffer preparation with EC less than 0.5mS. I will be very grateful if someone can help me to prepare this buffer.
If you're using HIC, why are you varying pH and not varying hydrophobicity?
Also, by washing with water you reduce the hydrophobicity.
You should use your binding buffer to wash the column, not water.
Mostafa Khedrinia added an answer
21 seconds ago
I have a mistake in my question. My Elution buffer's EC is 1.5 mS. I use hydrophobicity pH to elute my protein.
I use water to wash colomn after Elution.
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