I should have one peak of stained platelets. I am staining an intracellular protein after fixing and permeabilising. I keep getting two peaks in various samples, as if some of the platelets are not being stained. This makes it difficult to analyse data.
Double peaks may occur if there is insufficient antibody present- have you performed a titration of the antibody you are using to stain?
The possible explanation i can think of:
1) The target protein expression low and high amount in two platelets subsets;
2) Considering the size of platelettes, some debris may included during the analysis which will produce another peak;
3) Some polyconal Ab contain low and high affinity Ig, and this is not very likely in monoclonal Abs.
Is it possible that some of the platelets have degranulated prior to fixation? What are you using for fixation and is it fresh? I would also recommend going back to your FSC and SSC plots and seeing if the phenotype segregation can be attributed to differences in size (FSC and /or granularity (SSC)
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