The general approach seems fine. Personally I would use lower pH (2.3) but you are fairly close. Is your anti-IgE Ab totally selective? Do you recover ANYTHING from the membrane? I'm thinking perhaps the IgE is being competed out by other Igs in the sample, hence the question about selectivity of the capture agent.

Hi Nick, thank you for your reply. We indeed recovered something from the membrane. We performed a non-reducing SDS-PAGE with some of the purified samples and appeared a band in 55kDa instead of 190kDa (image attached, the first 3 lanes are commercial IgEs used as controls and lane 4 and 5 are two purified samples). We performed a reducing SDS-PAGE as well and we obtained the same 55kDa bands.

We are using the IMMUNOCAP TOTAL IgE column for purifying... I assumed is totally selective?

Thanks for the gel. I am not familiar with the Immunocap products but the supplier should be able to confirm the selectivity. So there are two questions. What is the 55K product? Why is there no IgE? Any chance the 55K protein could be BSA used to block the membrane? (I know this seems a bit low for the molecular weight). This could be tested simply by 'eluting' a blocked membrane. If this is not the explanation and the 55K protein originates from the serum, I would check whether more stringent washing prevents its binding to the membrane. If not, then I'd ask the question whether it is a degradation product of IgE and captured by the affinity membrane. A western blot might be informative.

Thank you Nick. The supplier does not reveal too much information regarding their product but yes, it's suppose to be selective. We will probably perform a wester blot and see what results we get. Thanks again for your help!