I am experiencing issues with the expression of phosphorylated proteins in my western blot experiments. Specifically, I observe strong phosphorylated protein expression but no expression of the corresponding total proteins in the same samples. For example, I detect phosphorylated STAT1 (pSTAT1) but not total STAT1 protein. Similar results were obtained for pSTAT3 and STAT3. I have thoroughly searched online but have been unable to find a possible explanation for this phenomenon.
I would greatly appreciate any advice or suggestions from anyone who has encountered a similar issue.
Here is my experimental protocol:
Hi Miao Shan Lin
Have you run a control sample treated with phosphatase? If your protein is heavily phosphorylated, you can check the dephosphorylated state of your protein of interest by treating with Lambda protein phosphatase.
Best wishes.
Dear Satyendra Mondal, thank you for your advice! I appreciate your suggestion to run a control sample treated with phosphatase. This additional experiment will help determine if the observed phosphorylated protein expression is specific or if it could be attributed to non-specific binding or other factors. I will definitely incorporate the phosphatase treatment into my experimental setup to validate the specificity of the phosphorylated protein signal.
Furthermore, I think I will revisit my stripping strategy to ensure its effectiveness in completely removing the previously detected phosphorylated proteins. It is crucial to have a robust stripping method to ensure that subsequent detection of total proteins is not influenced by any remaining signal from the phosphorylated proteins.
Thank you once again for your valuable input!
You might also try running separate, replicate gels, one for phospho- and one for total protein. Instead of stripping and reprobing.
Also, are you 100% confident in the specificity of the phospho antibodies? Is it possible the bands for pSTAT1 and/or pSTAT3 are really aomething else?
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