On 10 Jan 1997 jaensch at mpimg-berlin-dahlem.mpg.de wrote:
> I would like to purify a protein complex with the help of affinity
> chromatography. Therefore, I coupled an antibody, which recognizes one subunit
> to CNBR-Sepharose (Pharmacia).
> I have the following questions in this regard:
>> 1. Is it resonable to couple antibodys to CNBR-Sepharose?
This is pretty common. You could also use a crosslinker (NHS-Ester) to
link your antibody to beads. This has the advantage that you have a
spacer between your bead and the antibody which decreases steric hindrance.
>> 2. Which buffers are recommended for the affinity binding and for the elution
That depends on your protein. Typically people use 10-50mM Tris pH7-8, or
TBS or Hepes with about 150mM NaCl or KCl to decrease nonspecific binding.
you can also add glycerol to stabilize your protein. Detergents like
Tween and Triton-X help reducing non-specific binding.
Elution is usually done with 10mM Glycine pH 2.8-3 and immediate
neutralisation after elution. If you have a peptide
you can also elute by competing off your protein, which is more gentle ,
but leaves you with the problem of peptide removal. There is also a
buffer from Pierce that is supposed to be pretty gentle for elution,
hence the name Gentle buffer.
you might want to check out some literature, like "Antibodies, A
Laboratory Manual" by Ed Harlow and David Lane. Some companies (e.g
Pharmacia, Biorad, Pierce) also have pretty good handbooks.
Hope that helps
Toumy
