Dear all,
I checked my protein expression in the sphere cells through western blot. I could not see any expression. I added whole cell lysate and sphere cells. I could not see any expression of my protein. I want to know, How can I confirm my protein expression if I did not get any band in western blot and I got Beta-Actin band aswell.
Thank you
It could be a protein isolation problem (isolation buffer), How was protein estimation (concentration)??
If not then it could be a transfer problem, antibody not worked. How you developed the band??
If you provide a little more detail, then members can give better suggestions.
Did you transfect the cells with a plasmid with your gene of interest or do you use stable cells? Do you just want to see an endogenous protein being expressed?
How many cells do you use for your samples (from 6-well plates?)? How do you prepare your samples for the gel?
first validation of primary antibody and also origin use mouse or rabbit not human for best blot use sigma or invitrogen antbody originating from mouse or rabbit or goat.
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