Hi everyone,
I am a first year PhD student and I am working on a protein called Thymosin Beta 4 (TB4) and using it exogenously to treat injured cells in the kidney called podocytes. I am mainly working in cell culture right now and I am treating the podocytes with TB4 by adding it to the media that the cells are cultured in.
One recurring question I keep getting asked is how do I know that the TB4 is entering the cells? I have seen results when TB4 has been added, so I know that it is definitely having some sort of an effect. Does anyone have any idea of how I could prove that the TB4 is entering the podocytes? Any help would be greatly appreciated.
Your interest is to confirm the localization of you protein - have you consider fusing your protein with a flourescene protein reporter , then examine the location of flourescence. Unless if your reporter could significantly impact on translocation of the said protein another idea is if TB4 has a unique activity can you treat your cells from culture so that they are free from exogenous TB4 and then prove TB4 activity
Hi there,
Some experiments to think about:
Immunofluorescence using specific antibodies and intracellular markers (colocalization)
Increased resistance to protease degradation during the cell treatment
Neutralizing the cellular effect by preincubating the protein with a specific antibody (neutralizing antibody)
Hi guys,
Thanks for the suggestions. With these assays, since TB4 is expressed naturally quite highly in most cell types, I'm not sure if immunofluorescence would work, as the Abs would bind to the endogenous Tb4.
Hi again, if the exogenously added TB4 is somewhat different from the endogenous (tagged, labeled with a fluorescent probe) you might still consider localization experiment.
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