I am running a simple western experiment (using WES machine) where I test cytosolic versus nuclear fractions of mouse B cells (Using only 1million cells) and unfortunately Tubulin signal is not good enough to use for normalising the data. So I am looking for an alternative good cytosolic marker and a loading control.
I appreciate your help so much.
Alpha tubulin should be strictly cytoplasmic, while LaminA/C is a good marker for nuclear extracts.
If you observed an alpha-tubulin signal in the nuclear fraction (I guess it's the reason why you said it's not good enough), it is likely that the fractionation procedure was partial. This is a classical issue, since microtubules and ER are stuck to the nuclear envelope. If the fractionation procedure is too mild, cytoplasm contaminates nuclei, and if it's too harsh, some nuclei can be broken and contaminate the cytoplasm.
i would recommend using HPRT as your cytosolic marker and loading control. antibodies are extremely robust and yield very good signal. in parallel, use Histon H3 as your nuclear marker.
Best of luck!
Agnieszka Bojko Thanks a lot :)
Philippe Fort Thanks, but I am using Thermo Kit that states a 10% contamination.
Peter Bou Dib Thanks a lot Peter, I will give a try.
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