"Artem Evdokimov" <AEVDOKIMOZ at cinci.rr.com> wrote:
>The best thing to do if you never did this before would be to start with a
>decent book on affinity chromatography. If you are in a hurry, look at
>(e.g.) Pharmacia's web site - they have several kinds of pre-activated
>resins available for different applications. Historically, CNBr activated
>resins are used, but beware - these tend to leak. The coupling itself is a
>piece of cake, and is sufficiently covered even in the notes supplied by the
>manufacturer with the resin, but again - if you are unsure, read a book.
>>www.protocol-online.net/immuno/antibody/antibody_purification.htm
What he said :-)
Additionally, since you are going to elute with the antigen, non-covalent
"coupling" is also an option. Protein A and Protein G agaroses are sold
by nearly every company. In this case you first bind your antibodies,
wash rigorously (up to 0.5 M salt, detergents - OK) and load your
sample. Affinity for IgG is high enough not to worry about leaking of
significant amounts of IgG together with your protein. (You can always
crosslink them to Protein A/G though). Since Ab are from rabbit, there is
no real advantage of G over A while G is more expensive.
DK
>A.G.E.
>>"soenke behrends" <behrends at plexus.uke.uni-hamburg.de> wrote in message
>news:9tlsfh$5d8$2 at rzsun03.rrz.uni-hamburg.de...>>>> Subject: peptide antibody immobilization on a column
>>>> Dear netters,
>>>> I would like to immobilize peptide antibodies (raised
>> in rabbits) to a column for immunoaffinity purification
>> of a protein. I would elute with the peptide used for
>> immunization.
>>>> Do you have a good working protocol or can you advise
>> me on a material, kit, pre-packed column etc. from a
>> specific supplier that works particularly well for
>> that purpose?
>>>> Thanks for your time and help
>> Soenke
>>>>>>
