Hello all! I hope that I am posting this in the correct place. This is my first post here! Hopefully someone has some suggestions/critiques that can help me out. I mostly would just like to see if anyone else knows of good, alternative ways to purify and isolate parasite (or other organisms) secreted proteins bound in host immune complexes.
I first centrifuge the whole serum through 5% PEG-6000 at 3000RPM followed by two additional washes with 2.5% PEG-6000 to remove unwanted serum components. The pellet is then resuspended in PBS buffer. I have seen this protocol in a few papers, and it seems to fit for the purposes of my project.
Next, the resuspended sample is put through an affinity column with Protein A sepharose beads. My thinking is that the IgGs that are bound in immune complexes will stick to the protein A beads and the whole complex will stay on the column during subsequent washes.
I then elute with a strong acid (pH 2.8) and collect fractions. I check the OD280 NanoDrop reading for each fraction to see when the immune complex proteins come off the column, before pooling the fractions and concentrating the sample.
I believe that the sample could then be run on a SDS-PAGE gel without any reducing agents. This would theoretically keep all of the, now free, whole antibodies from entering the gel due to the unbroken disulfide bonds within each antibody. The molecular weight would be to high for them to run with the proteins of interest.
The ultimate goal of my experiment here is to send the collected proteins out for mass spectrometry, to identify which proteins can be found in vivo. The problem is that I just sent my sample to have mass spec done and the results don't show any parasite proteins whatsoever. Does anyone have a suggestion on how I might go about purifying these proteins from serum to send for mass spec???