I want to analyze condensin binding pattern onto Drosophila Chromosomes (S2 cells), but I have a problem with the immunostaining; I've been trying to detect the chromosome-axial pattern that is supposed to be for condensin, but so far I couldn't see any protein staining at the chromosomes. I can see some staining in interphase nuclei and some cytoplasmic signal (although I cannot be sure whether this is background or not) but it is absent in condensed chromatin. I followed a protocol suggested by a paper in which they analyzed the same proteins in the same cell line, but I couldn't obtain the same results. So I've tried different Triton concentration in PBS for fixation and permeabilization, I've tried different TSA concentration for blocking, I've tried different concentrations of antibody, but the results are always the same.
I am not sure whether this is a problem of the protocol or a problem with the antibody itself. Western Blot shows the band at the expected molecular weight just for CAP-H and CAP-D2, but I couldn't detect it for CAP-H2 and CAP-D3 shows a clear band but with a different molecular weight (from expected). Immunostaining results are the same with purified and non-purified antibodies. So as you can see, I have had many problems with this products
so I wanted to ask you what do you consider to be the problem here and how can I be sure about it (some way to know that is the antibody and not the protocol that is wrong)
Thank you so much for your help!