Immunoaffinity chromatography - (May/11/2008 )

HI friends
I want to purify my protein using antibody affinity chromatography with antibody specific to my protein.
I am looking for protocol for making immunoaffinity column.One protocol says that use 0.2 M sodium borate for column coupling with antibody.
has anyone done this?please suggest me That can PBS be used in place of o.2 M sodium borate.As my Co IP experiement worked well with PBS for this particular antibody.

Thanks

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borate is the buffer used for coupling the antibody to the matrix. you would use different buffers when applying and eluting the sample.

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There are several activated coupling resins you can buy. I've had success with Affi-Gel (Biorad?) and CNBr-sepharose. They are quite simple to use and to estimate mg Ab per ml matrix.
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Thanks for the response
I have protein G sepharose from amersham.So i wanted to use it.I am just wondering if other than borate buffer any other buffer like PBS can be used for coupling.
If only borate buffer has to be used then cud u suggest me that whether its borate buffer saline or tris borate buffer.As its very confusing.coz protocol just say borate bufer without any specification.

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I think that it depends on what compound you are using to couple the antibody, but in general I think that you are supposed to stay away from anything that has amines during the coupling step. In the end, you can resuspend your antibody-coupled beads in PBS.

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since you are binding to protein g, you can use pbs. it is a simple binding. but, if you want to crosslink to stabilize and make permanent the binding to protein g then you may still want to use borate.

the borate to which they are referring is probably just boric acid/sodium borate adjusted to the desired pH (you can take boric acid and adjust the pH with naoh or you can use equimolar boric acid and sodium borate and one or the other until the solution reaches the desired pH).

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Is this Protein G sepharose going to be used to covalently couple the antibody? If not, just use PBS or TBS. If it is an activated matrix for coupling antibodies, follow the Amersham instructions. But, if you want to make an antibody column, you really do not need protein G. Protein G sepharose is a great affinity column to purify certain antibodies, but it is not routinely used to make antibody-affinity columns (I don't think).

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protein a and g are used to couple antibodies by the tail so that all of the bound antibodies will be available to bind antigen, unlike general binding gels (eg cnbr, ch or ah-sepharose) which will bind the antibodies with random orientation.

pierce has a kit (originally designed or, at least, sold by s&s) that uses protein a or g and crosslinking to make stable immunoaffinity gels with properly oriented antibodies.

see this link:

protein a and g orientation kits

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