I have used B-actin as a loading control for cell proliferation in Western blot against the treatment of a bacterial extracellular extract. The intensity of bands is not equal in all lanes, so, I'm just wondering is B-actin act as an appropriate loading control for cell proliferation. I'm using PCNA as a cell proleferation marker.
Hi
You must be specific about your extraction buffer (lysis buffer) and the method used for protein quantification.
my first guess is that equal amounts of protein were not loaded into each well. Detergents are not compatible with bradford reagent and BCA method interfered with antioxidants. The best choice is to provide high concentration lysate and then diute them to lesser the antioxidant disruption. Then, BCA would be the choice.
Best
In addition to that, you will oftentimes see that certain loading controls just won't work under certain experimental conditions or with certain cells. See this post:
https://www.researchgate.net/post/Housekeeping_proteins_loading_controls_for_a_Western_Blot_of_E_coli_cell_lysates2
It helps to try different genes as loading controls and look through the literature (i.e. which controls other people used successfully in the past).
Recently born cells can be immunolabeled for PCNA, so there is some margin of error in using PCNA as a protein marker for rates of proliferation. As for loading controls, I suggest you take a look at the following publication:
PLoS One. 2012;7(8):e43028. doi: 10.1371/journal.pone.0043028. Epub 2012 Aug 20.
Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development.
Rocha-Martins M1, Njaine B, Silveira MS.
Hi Sandeep , beta actin is not the correct loading control for proliferation. The best control for proliferation would be Ki67 levels not even PCNA. You can also use H3S10p as a control if your study aims to differentiate between mitosis and G1 phase. You can also use cyclin B