Dear all,
When I run my yeast total protein lysate extracted after 10 minute-treatment with my compound, I observe a global up regulation in proteins, even my loading control protein. I suspect the compound affects extraction/cell membrane permeability etc, therefore treated samples show up regulation in multiple proteins apart from my target protein as if they are not loaded evenly. Before running my samples, I measure protein concentration with BCA. I repeated many times, but the outcome is always same. I need to show that my target protein is upregulated regardless of the background. How can I bypass the issue? Would spiking a recombinant protein be helpful? Is there anyone faced with a similar problem? Thanks
Regards,
Burak
And https://www.frontiersin.org/articles/431886
After doing the protein assay to measure the protein concentration in each sample, run the same amount of total protein (in µg) for all samples on the gel. The amount of control protein should therefore be essentially the same in all lanes even if the extraction efficiency differs for each treatment. Then you can measure the ratio of the each protein band to the control band.
So you have measured the protein concentration? How much protein did you load per line? Did you load the same volume or protein amount? Because you must load the same amount of protein as Adam wrote, but what you wrote sounds like you did that already.
Be aware that the amount of control protein all lanes must be similar
Thanks all for your answers. I load equal amount of protein and equal volume. However, my loading control proteins still differ in band size sometimes. The treatment duration is just for 10 minutes
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