Im seeing immunomodulation and antioxidant activities of my plant methanolic extract fractioned into chloroform, ethyl accetate, butanol and aquoeous.
It is good question. Usually, more than one protein loading control is desirable to compare different human cell lines and primary cells. I would recommend beta-actin and tubulin. You can find more information in the link from Bio-Rad.
Link: http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6360.pdf
it is really very good question and i agree with the answer of Divaker Choubey.
but I want to add something here. if you are talking about the loading control in cell line then you must decide according to molecular weight of your protein of interest.
Please select one of the three commonly used loading controls (beta-actin, beta-tubulin and GAPDH) based on mol. wt. of the protein you are interested in.
Depends on your experiment. These days, the best recommendation is to maybe even use whole protein lysate deriving from Ponceau staining as control. Depending on cell type, every protein might be differentially expressed. Beta-Actin, GAPDH and tubulin are commonly used, but depending on cell type and condition might differ a lot.
While you want to compare expression of some proteins in different cell lines, the type of the cell lines should be taken in to the account. If their histological origin are different, selection of more than one control is recommended.
However, comparison of results of several cell lines should be interpreted very cautiously due to easily misleading conclusions.
I'd compare total protein content using membrane staining with ponceau or any other protein dye. Any specific refernce gene may vary between cell types.
Beta-tubulin is one of the most commonly used reference loading controls for western blotting. It is due to the fact that is abundantly expressed across various tissues and developmental stages and also highly conserved across species. However, since there has been observed some variability in expression levels of commonly used housekeeping genes (PMID: 15627964), I would suggest using more than one loading control antibody. Some further suggestions control antibodies can be found here: https://www.ptglab.com/news/blog/loading-control-antibodies-for-western-blotting/.
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