Affinity purification of antibody - (Aug/03/2007 )

Hi,

We have received some antisera (rabbit) generated by immunizing with some relatively small peptides (11-15 amino acids). We have purified IgG from these sera, but they do not work very well in immunohistochemistry or immuno precipitations (much unspecific binding). So, we need to improve them, and we want to do affinity purification since we have all the peptides that were used for the immunizations. However, we don't have any experience with affinity purification and it seems to be a jungle of possible protocols out there... Does anybody have any recommandations? Any protocols that are relatively easy to use? For instance, we don't have a pump so we have to depend on gravity or a syringe when washing/eluting.

Any help is greatly appreciated!

Regards,
Sonja

Did you test Ab titer by ELISA against your peptides? May be first of all you should not improve method of Ab purification but design of antigen ( as I understand peptides are epitopes of protein). So there are two possible problems may be - work with epitope design and improve scheme of immunisation ( may be immunogenicity of your peptides are low)

For preparation affinity column you need near 10-15mg peptide per 1 ml resin. If you have little experience with coupling ligands for aff columns it will be better to buy preactivated matrix ( BrCN, or epoxy-activated) ( Amersham, for example) which contain active groups suitable for one step conjugation of peptides.

Hi,

The producer of the antisera has tested them by ELISA, and the titers were evaluated as "good". Regarding the design of antigen, we don't have much choise; we are working with splice variants of a receptor and the exon specific for this particular splice variant is very short, so there wasn't many peptide sequences to choose from... Regarding the immunisation protocol that has been used, we are totally dependent that this firm know what they are doing (Genosphere biotechnologies). I hope so. They should, since these antisera are very expensive...
We were thinking to use preactivated columns of some sort, but even after deciding that, I think there is still a lot of different things to choose from (CNBr, epoxy, NHS etc...). I was kind of hoping that someone had tried different alternatives here and perhaps found that some were better than others for short peptides, or something like that...

Regards,
Sonja