I am doing westernblots. Thee independent experiments. In each experiment i get different result. For example, if i see a decrease in my target protein at 10ng/ml then in another repetition, it is decreasing at 5ng/ml.
I have optimised by dilutions. I am confused what could be the reason behind it? Is it the health of the cells?
Your question is confusing. What is your purpose for western blotting? Are you analyzing protein concentration with western blotting or you are using the same concentration of protein sample but in the results of the western blot you are getting protein bands with different thicknesses? If it is possible for you to clarify your query we can troubleshoot the problem.
My question is Usha Kantiwal i know that when I treat cells with a drug, it downregulates my target protein. I have already done a pilot experiment for that.
Now, are doing a concentration dependent study and when I am doing independent experiments to generate good data, I am not getting consistent results.
for example, in one set of experiment, I see complete downregulation at 20ng/ml whereas in another set of experiments the complete down regulation is at 40ng/ml.
2. another issue I am facing is with the increase in concentration of drug, there should be a linear decrease in my target protein on western blot. However, I find in some of my blot, a much thicker band at 1ng/ml drug concentration than 0.5ng/ml.
these two things are happening repeatedly. I am not able to find where the problem is, is it the problem of transfer of proteins, or the cells, or protein quantification or antibody
could you help me troubleshoot this?
Are you using the same protein aliquot for the inhibition study? If yes, protein concentration should not be an issue but if you are using different aliquots protein concentration might be an issue. And if you are using samples with different drug concentrations on the same blot then the transfer of protein should not be an issue. But it is not necessary that you will get a linear decrease in protein with increasing concentrations of the drug. The difference of 0.5ng/ml and 1ng/ml is less so you can try the inhibition assay with larger drug concentration differences. You can eventually move to decreased drug concentration differences once you get better results with higher drug concentration differences. Have you tested the results with SDS-PAGE? Are you getting the desired results there? You should optimize the results with SDS-PAGE first then move to western blotting. If you are getting good results on SDS-PAGE and not getting the results on western blot then we can say that there is an issue with the western blot procedure. I hope my answer will be helpful to you. Thank you!
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