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affinity purification of polyclonal antisera

affinity purification of polyclonal antisera - Protein Forum

affinity purification of polyclonal antisera - Protein Forum


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  #1  
Old 08-09-2006, 12:13 PM
liss
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Default affinity purification of polyclonal antisera



I am in the process of affinity purifying polyclonal antisera we made
to make it more specific to our desired protein. I have purified by
nickel affinity the protein we are going to use to anchor the
antibodies but I need to find a kit or something that will result in
good yields, please let me know if anyone has any ideas on the best
methods or kits to use?

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Old 08-09-2006, 01:12 PM
StewJW
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Default affinity purification of polyclonal antisera

What kit are you refering to?

If you need to bind your protein to a solid matix for affinity
purification the easiest method is to use CNBr activated sepharose.

[Only registered users see links. ]

The Amersham fast flow is better but the 4B version maybe cheaper and
adequate for small scale.

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Old 08-09-2006, 08:06 PM
DK
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Default affinity purification of polyclonal antisera

In article <1155125612.114466.125920@h48g2000cwc.googlegroups .com>, "liss" <[Only registered users see links. ]> wrote:

As another poster suggested, no kit is necessary. My preference
would be NHS ester activation over CNBr (mostly due to the stability
of the linkage). IMHO, you can't go wrong if you buy Bio-Rad's
mixture of Affi-Gel 10 and 15 and follow their protocol.

Before you purify in the antigen, please do yourself a favour
and first isolate IgG fraction from your antiserum and use that to
purify on immobilized antigen. Your results (particularly if you intend
using the antibody in any kind of response inhibition study) will
be sooooo much cleaner ans trustable if you do.

DK
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