Hi, I am new to this platform. I am currently preparing for Western Blot experiments using Young (7-10 mo) and Aging (20+ mo) mice and the protein of interest will be tight junction proteins (ex. occluden, claudin-5, ZO-1).
I have looked through a series of commonly used housekeeping genes (e.g. β-actin, α-tubulin, β-tubulin and GAPDH) but found that all of them will be affected by aging. I have also looked through stain-free and total protein normalization (by Ponceau S or Coomassie staining), which seems to give a more promising result than the housekeeping genes. But since they are relatively new approaches, I would like to seek opinions here about a good way for the loading control of age-related Western Blot.
Thank you very much!
You can refer to the article:Article GAPDH and -actin protein decreases with aging making Stain-F...
Hello Hongqian Wang
You may use Rplp0 (ribosomal protein large P0).
You may want to refer to the article attached below which has shown that out of the eight reference genes examined (Gapdh, Gusb, Rplp0, B2m, Tubb5, Rpl7l1, Hprt, Rer1), Rplp0 was stable in both sex as well as age.
https://www.aging-us.com/article/204710/pdf
Best.
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