IP for ceruloplasmin
I want to deplete 15 mL of human and sheep plasma for ceruloplasmin.
I have been looking up methods but I'm confused about how do go about it.
Most people seem to follow this protocol: incubate plasma with an antibody raised against ceruloplasmin. Add IgA and/or IgG conjugated agarose beads. Incubate and pellet the beads.
But for 15 mL of plasma I would need a lot of expensive antibody.
Is there a protocol and/or product anyone can recommend? A bead system, or maybe an affinity column I can load with a specific antibody, another method? Or recommend where I should be looking for these answers?
Thank you for any suggestions.
Last edited by kvrancken; 08-21-2009 at 03:57 PM.
Re: IP for ceruloplasmin
Preparation of ceruloplasmin-depleted plasma
Ceruloplasmin is immunoprecipitated from human plasma by treatment with rabbit anti-human ceruloplasmin antiserum in 0.05 M TES buffer at pH 7.3 and incubating for 2 h at room temperature, followed by incubation with protein G-agarose in 0.05 M TES buffer at pH 7.3 for 2 h and centrifugation. The ceruloplasmin-deficient supernatant is concentrated by ultrafiltration using Centricon-10 filters (Amicon, Beverly, MA) and stored at -20 °C. Complete removal of the ceruloplasmin is verified by SDS-PAGE on a 4-12% polyacrylamide gel followed by western blotting with rabbit anti-human ceruloplasmin antiserum.