I'm trying different protein extraction methods and detection to be able to detect cyclins using western blotting, but I have not been successful so far. The last method I tried was TCA precipitation. My control proteins (MCM6 for instance) is beautiful on western....but cyclins has been a problem..... Any input would be greatly appreciated!!!! :)
If your doing in vitro, what kind of cells are you using? you may need to serum starve the cells and synchronize them by giving growth factor for a certain amount of time (which will depend on cell type and which cyclin you want to observe).
could also be a problem with the antibody - cell signaling has a cyclin D1 antibody that works beautifully for western.
Thank you so much for your comments Patrick! We are trying to detect cyclins on Tetrahymena thermophila, to learn more about its replication. We do have a way to synchronize the cells using elutriation process. I will also look at the D1 antibody you mentioned. The main cyclin we are interested is Cyclin 22, a cyclin D family.
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