Hello, I am evaluating the expression pattern of some proteins during the process of neuronal differentiation (1 day in vitro to 32 days in vitro) in primary neuron culture using western blot. I tried to normalized the results using housekeeping proteins such as b-acting. However, the housekeeping proteins time dependently increased during this period so I cannot use them as loading controls.
How can I normalized my WB results?
Thanks
Hi!
Alternative methods for you.
There is a total protein normalization procedure with ponceau staining. Look for the question in research gate: Can Ponceau staining of membranes be used as a loading control ?
Also, if you have money and the equipaments, try stain free normalization. Article: Stain Free Total Protein Staining is a Superior Loading Control to β-Actin for Western Blots.
Good luck!
Jacó
I agree with Jaco, the total protein stains may be a suitable method. However, if your lab has access to common loading controls, you can also try these proteins:
https://www.abcam.com/primary-antibodies/loading-control-guide
Alejandra Llanes-Cuesta May be in your case beta-actin is not an appropriate loading control. You can try with other housekeeping genes to confirm it. It is a usual practice to include more number of loading controls to check consistency and credibility of western blot data.
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