How can i enrich highly -ve charge biomolecules using High Q resin? - (Dec/23/2014 )

Hi All,

I am working with very special protein,which is modified by several number of sialic acids(-ve charge).Some how i am finding difficulties in purifying protein of interest by affinity.So, i am trying to at least enrich -vely charged molecules and to see weather i can get my protein of interest in the fractions.

This is my first GO!

I am using Macro prep high Q anion exchange resin for enrichment.experimental set up is biorad column with pump.My buffer A composition is 50mM tris HCl, 100mM NaCl and1mM EDTA. I am planning to do Step elutions with buffer A containing 0.2M, 0.5M, 1M and 1.5M NaCl.

Now My question is:

Is it right buffer composition what i am planning to use?

My native protein has pI of 9.4 but modified protein get more -ve charge protein may behaves differently.What pH of buffer may i use?

I am doing step elutions as my sample has many other proteins,So, Which salt concentration i can use to elute more -vely charged proteins?

Plan is to get rid of low  -ve charge molecules at lower salt concentration and get enrich protein with high -ve charge at higher salt concentration!

Thank you for you help

Wish you Marry Christmas

Som

what pH are you planning to use? if around neutral then you may want to use a cation exchange resin.

you can also elute with a pH gradient.