Hi all
I am doing an experiment whereby I extract IgG from serum using an affinity collumn assay (Protein A- columns: NAb Spin Columns, Thermo Scientific) , and I want to expose cultured endothelial cells to the negative fraction of serum (IgG-depleted serum).
However, per protocol the serum has to be diluted (1:4) before passing through the Protein A- columns (NAb Spin Columns, Thermo Scientific), and the effect I am looking for is lost when cells are exposed to the diluted serum.
So two choices:
a) I try to pass undiluted serum through the columns
b) I try to "re-concentrate" the negative fraction through dialysis with a concentrating gradient (such as Spectra/Gel Absorbent with FLoat-a-Lyzer G2, Spectrum Labs).
Do you have any experience with any one of these alternatives?
Best,
From my point of view, the first choice is preferable. The reasons for the dilution are the following: Reduction of the viscosity. If you do not dilute the serum, the binding to the column will be slower. Hence, the flow rate should be reduced to compensate for this or repeat the depletion step until the IgG level is low enough. In addition, a lower concentration will reduce the effect of non-specific binding (NSB), particularly of albumin and other highly abundant proteins. This seems to be less relevant for your depletion experiment. BTW: You should consider that Protein A does not bind all IgG classes with the same efficiency.
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