Hello, I have been working with a polyclonal antibody (goat) raised using a recombinant protein (From R&D systems); the sequence corresponds to the EC domain of the protein I am looking for. The secondary antibody is an anti-goat conjugated with PE. I have also been using a recombinant protein for detection with a human FC tag, and an anti-human FC FITC conjugated antibody. Various cell lines have been tested and analyzed with flow cytometry.
My problem is that the antibody shows close to no binding; however, once we fix (4%PFA) the cells, the binding increased from 2-3 % to 60-70% in terms of positive cells. We have also tested a recombinant protein for the target receptor with low binding on viable cells and high on fixed cells. The binding of the antibody and the recombinant protein have been tested with various temperatures, incubation times, and concentrations. With a low % of positive cells in all cases for viable cells.
So my question is, why does the antibody/recombinant protein bind to fixed cells and not to viable cells. Could it be that the fixating causes the antibody and the recombinant protein to bind unspecific to the cells, and if so, how can I be sure that this is not the case?
I really appreciate any help you can provide.
Did you run your samples in buffer with azide at 4oC? If not, the receptor might be modulating off the cell surface of your viable cells by shedding or internalization, but does not when they are fixed. I see this looking at mouse CSF-1 receptor on RAW 264.7 cells. This is particularly true with polyclonal antibodies, but it can be true of monoclonal antibodies as well.
We have even used fixed RAW cells for a competitive 'RIA' with I125-CSF-1 as the label to test the CSF-1 content of samples in the past, so in our case, fixation made CSF-1 receptor available for the tests. Sometimes, fixation destroys binding, in our case, it stabilized it.
Gary Lee Gilmore Thanks for the fast response. I will try this next week.
Thomas Sjöberg Another possibility is the 3D structure/flexibility of the epitope in intact cells when anchored in the membrane, which might be different from the 3D structure of the recombinant immunogen. Do you have any information how the antigen has been produced (expressed in bacteria or mammalian cells, denaturing purification conditions) and about the 3D structure of the protein you're after?
Hello Jamie Cooper
, according to the manufacture, the epitope is part of the EC domain of the surface protein. We are fixing it with 4 % PFA for 15 minutes in RT. We have tried with shorter and longer times in 37C, RT, and 4C, not affecting the results.Wolfgang Schechinger The protein was derived from Mouse myeloma cell line NS0, can't find any information about the purification conditions will try to look in to this further.
Thank you both for the answers
Thomas Sjöberg As Phycoerythrin is a rather large molecule and thus PE conjugates, could it be that the secondary does not find its target? Do you have a secondary that has been labeled with a rather small reporter (e.g. fluorescin) to check?
Hej Tomas. We developed a technology to monitor binding to cellular receptors over time and for this real-time data extract the affinity and binding rate constants. This LigandTracer technology is used by many Ab developers in addition to FACS and one of the reasons is that it readily shows processes that are more than just simple binding, e.g. multiple affinities, receptor shedding and internalization mentioned earlier. We are currently working on more exact internalization models to extract the internalization rate relative to the amount bound. You are welcome to try this technology (has both PE and FITC compatible detectors).
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