We have a knockdown THP-1 cell line that has cytoskeleton and structural proteins affected. We are doing western blots and tried using Actin, Lamin-B and GAPDH as a control but their expression changes.
Can anyone suggest an alternative?
Do you have any ideas which cellular functions could be stable? If you influence the cytoskeleton proteins such as actin or tubulin should be ruled out. If mitochondrial functions aren't influenced you could try citrate synthase or glucose metabolism enzymes. Some heat shock proteins can work as well.
Margit might be onto sth, i.e. your loading might not be equal in the first place. I normally use ponceau stain every time I run a WB, followed by Ab staining. If you want another housekeeping gene, we frequently use Hsc70:
https://www.scbt.com/scbt/product/hsc-70-antibody-b-6?requestFrom=search
Hello Ines,
I hope you find this chart helpful: http://www.abcam.com/primary-antibodies/loading-control-guide
All the best!
I always use Ponceau staining first to check for blot efficiency and to see if I had any air bubbles in the blot (edit: because if Ponceau reveals a bubble where a relevant band should appear you can often save the trouble of AB incubation and repeat the blot). You can estimate if protein loading is equal but this should be taken with a grain of salt in my opinion. Often times you see differences in your housekeeper that weren't really distinguishable from Ponceau staining. You also have to wash it thoroughly until your de-staining solution is almost clear.
However I find that Ponceau staining is an invaluable quick check that should be performed with every single blot. It only takes a few minutes and costs basically nothing.
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