I would like to isolate the IP products using human serum as a source of antibodies. Several papers report the use of sepharose 6b columns to fractionate the IP products. I am looking for a quick group separation approach (similar to desalting). Can anyone provide the column dimensions and bed volume to use for this purpose?
When co-fractionating serum with your sample of interest by gel filtration, you might get a lot of unwanted contaminations in your fractions.
Have you considered
1) capturing IgGs from serum on immobilized Protein G (eg 10mg protein G on 1ml NHS activated sepharose)
2) eluting the IgGs and covalently coupling them to a resin (eg NHS activated sepharose, again 1 ml happily will do)
3) using this column for affinity chromatography of your samples?
Advantage of this approach is that you may process a lot of samples (even automated by HPLC or FPLC) with the same antibody affinity column. Saves lots of time, materials and resources.
Thanks Wolfgang,I was planning on the same lines. but I have to do a Mud-Pit proteomics at the end, and glycine-HCl does not works out very well for this purpose. My previous attempt of using 10mM HCl were not good to elute out the proteins. as for the co fractionation of unwanted proteins that cann be easily controlled by fractionating the tissue extract and the serum source separately and then do the MS for all three of them remove the common proteins between three samples
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