hi,

does anyone know about the purification of HRP antibody conjugate... I tried it with sephacryl gel filteration column .... but itz not giving good yield and conjugate is also in polymerized form. if anyone knows abt it.. plz let me know.....

Thanks in advance.

pallavi

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question: why do you want to purify conjugated antibody? normally antibody is purified before conjugation. or?

maybe it´s possible to purify with an affinitycolumn (with coupled peptide the antibody is against to).

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Could the polymerized form be due to conjugation reaction step, rather than a separation issue?

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to purify it from unconjugated ab and hrp. otherwise it will give a high background.
i tried it with con A affinity column also... but it didnt work out.

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this might be the possibility...

m activating the HRP by sodium periodate and mixing it with antibody for conjugation.
but something is going wrong becoz m getting the conjugate in the void volume of sephacryl gel filteration column..

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which sephacryl are you using?

the conjugate may be too large for s-100 or s-200.

maybe you should try s-300 (if you are not already).

what is the medium with which you are equilibrating the column?

do you have the proper ionic strength?

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i used s 300 and 50 mM PBS pH 7.2 as an equilibration buffer

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you may want to increase the ionic strength of your buffer (eg-150mM NaCl).

you can check if it is aggregated on native page.

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i checked it on SDS page. but it is not entering the page.

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Redo the conjugation reaction with a right ratio of HRP-[ox] and antibody. Its too late.

Conjugation is covalent in nature, you cant reverse it, and polymers are too large in sizes.

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