Dear all, I recently had trouble analyzing my data by Western blot. I'm using the cell line model and transfection performance to analyze the importance of my target protein to cellular signaling. When I transfected and visualized the protein location or cell vibration, migration, or proliferation, the results turned out as expectation. However, when I tried to extract the protein for western blot and RNA for q-PCR analysis, the data became inconsistent and the phosphor form was the same between all conditions. I had changed my sample buffer, lysis buffer also other buffers to make SDS-PAGE but the results were the same.
Could you please give me suggestions to solve the trouble?
Thank you so much and best regards.
What are your controls for both experiments? Could it be possible your transfection created artificial results due to transcription burden?
I don't think qPCR can quantify phosphorylation. Have you used phosphpo-inhibitor in your proteinase inhibitor cocktail? Some cocktails do not have phospho-inhihbitor in them. Also make sure to run your experiment as soon as possible to prevent protein degadation.
What buffer did you use to wash your western blot? Milk contains phosphorous and will bias your results, so you need to use BSA or similar.
Good luck!
Maybe you are sampling in a late time point, and the phosporilation occurs at early stage, try to do a time lapse experiment, as early as posible 0.5-2 h
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