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Protein Purification - Advice - dialysis/desalting/concnetrating and antibody production? (Nov/08/2005 )

Hello all,

I am new to the world of proteins and am trying to find my way around them.

I have expressed and purfied recombinant His-Tagged protein in E. coli. After this degree of purification I have 600ul of a 0.5mg/ml protein solution. What I would like to know is:-

1. How should I desalt this volume of solution? I want to do DNA/binding studies with my protein so the stuff in my elution buffer needs to be removed.

2. I have been making cell free extracts from mycobacteria - these are grown in medium containing tween. I run the dfx on an sds gel and they look good but when I come to quant them using the better bradford assay kit from Pierce I get a white pcpt. Is this because there is left over tween. How can I remove detergent from cfx?

3. I would like to use my recombinant his-tag protein to raise antibody to do westerns. How shall I purify it for this purpose?

many many thanks!


I can help you with the 1st question! Pierce makes something called a 'slide-a-lyzer', easiest dialysis method you'll ever use that results in very high purity.

I think you would have to remove endotoxin before you could make antibodies, but it's pretty darn pure stuff. if you want to concentrate it afterwards, amicon (or millipore? both?) make some little centricon concentration units that are quite effective and easy to use

good luck