Hi all,
I encountered an issue with my western blot : the loading controls I tried were all down regulated after 24h TGFb1 stimulation in NRK49F cells (rat kidney fibroblasts) so I can't use the results I got for quantification.
I tried b actin, alpha tubulin and GAPDH (see pictures)
Any ideas on what loading ctl I should use?
Data :
-10 % acrylamide gel in denatured conditions
-primary antibody 1/10,000 dilution in 5% milk TBST overnight at 4C
-secondary 1/20,000 dilution 1h/RT in 5% milk TBST
PS: the individual treatments are lipoxin based (anti inflammatory molecules) so they should not modify the expression of proteins tested, TGF is the issue here.
Many thanks!
Mariam
I would not say that the control is downregulated. In my opinion bith proteins (control and protein of interest are NOT regulated in this experimental setting).
I would propose to reevaluate protein concentration - maybe not via BCA assay (results of Bradford-methods are affectd by detergences in the sample buffer during measurement) - rather via a Lowry-based method (excluding the effect of detergences). This will lead to more "correct" values-. After that you should repeat the Western blot and corresponding analyses. In my opinion, this will result in an other outcome and maybe more reliable data.
Mariam,
It seems there are a lot of literature on your experimental model. Most of them have used GAPDH as housekeeping protein, and their house keeping protein seems pretty much even.
I do not question about your techniques. You can try re-quantification of your samples as suggested above. We usually use dilute primary antibodies 1:1000 in 5% milk TBST (Secondary antibody also the same). I feel your antibody dilution is too high (1:10,000 and 1:20,000). I would rather give a try with lower dilutions.
Cheers,
Susara
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