Hi All,
My project needs some streptavidin-oligo and antibody-oligo conjugates. But usually I could only use 20-60 ug antibody to do the conjugation reaction as the primary antibodies are usually very expensive. Then how can I purify the conjugates after the reaction from the unreacted oligo and antibody? I think size exclusion chromatography is hard to get a signal on that amount? Could you guys please give me some suggestions on that? Thanks a lot.
Best,
Gang
Very small gel filtration centrifugal desalting columns can be used for very low-volume samples to remove low molecular weight substances from large proteins:
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006051B.pdf
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-dialysis-desalting-concentration/zeba-desalting-products.html
Reverse-phase HPLC on a C18 column, monitoring at 260/210 nm, should also be able to accomplish this separation. For oligo conjugates I normally use triethylammonium acetate buffer / acetonitrile as the mobile phase ramping slowly from 0 to 75% acetonitrile over 45-60 minutes.
We use the biospin column (P-6 and P-30) from biorad to purify the Antibody conjugates, it work good but just follow the protocol recommended by the company. As we tried to modify it and found to have low volume recovery.
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