Hello,
I am doing western blotting from Human monocyte derived dendritic cells, differentiated in presence or absence of compound that inhibits the differentiation. I have tried using actin as loading control but I am not getting actin levels equal in both the samples. I am looking for phosphorylated protein and levels of total protein is also varying between the samples, so in such case, what loading control is used.
Thanking you,
Hi Komal -- have you performed a BCA assay to ensure each lane is loaded with an equivalent amount of protein? If so, and the actin still isn't equivalent between the two, GAPDH and Tubulin may be useful, whole-cell alternative loading controls.
Hi, Komal.
If you are getting unequal levels of actin despite the efforts to adjust protein contents after measuring the protein contents by Bradford assay, and equal sample loading, the cells treated with chemical need to be checked for cell viability with trypan blue staining to evaluate cellular activity at high concentration of the chemical. Dead or dying cells may give some total protein contents, but not a similar level of actin. Check it out.
Hi,
@Meaghan Roy-O'Reilly,
I had performed BCA and loaded equal amount of protein from both the samples.
I have read somewhere that actin levels, do vary on differentiation.
So i had thought that total protein levels for that phospho protein, will serve as loading controls, as used by others in similar study, but in my case I am getting even total protein levels varying between the samples.
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