I am working with phospho protein in western blot and have problems about the phospho protein signal.
It is common that researchers can not detect phospho-protein signal in the membrane, as protein phosphorylation is a fast process.
However, my issue is, I always have very strong phospho-protein signal in all of my samples, either control or the cells being treated.
My experimental condition are as follows:
1. Rest cell in serum free media for 16 hours.
2. Treat cell by treatments for 5 mins and wash with ice-cold PBS
3. RIPA (with proteinase and phosphase) lysis and protein qualification (BCA)
4. Dilute both primary and secondary antibodies (both are from Cell Signalling company, monoclonal).
I searched for many suggestions online and paid attention to each step as much as I can.
But I still have very strong phospho protein signal, here I attach a picture as an example. This is pSrc, in which all of the lanes show a similar pSrc protein expression. The first lane is my control, which should have no or very rare pSrc expression.
Does anyone have similar issue or could give me some advice? Many thanks!
Do you have a negative control, such as a mutant protein that cannot be phosphorylated? The purpose is to show that the Western signal is not non-specific background.
Since the negative control is still showing the band of interest just like all the other lanes, that makes me wonder if your primary antibody is also reacting with something other than pSrc. Fully read the datasheet for the antibody you're using and see if it lists other phospho-proteins that the antibody may cross-react with- that may give you an idea of what's going on. If that's the issue, you could try using an antibody from a different manufacturer that ideally doesn't list the same cross-re-activities. Also, if you have the time/resources, you could try probing for or inhibiting any of those unwanted proteins.
I'd also take a page from Adam B Shapiro's book and try using a different negative control- one that shouldn't contain a Src that can be phosphorylated.
Hope this helps! Good luck!
I agree with Adam, it looks more like background signal to me. I would suggest to run both negative and positive control to figure out what's going on. As a negative control you can also use a sample treated with buffer without the inhibitor that was treated with phosphatase instead. I also like to make my own orthovanadate and use that instead of the commercial inhibitors. Cheaper and better in my experience. As for the positive control, you can even buy one - though I prefer to make my own. Good pSrc primaries are hard to come by, so it is totally possible that you have a flawed antibody to begin with. I would recommend to try a different vendor for it or, better yet, look into something downstream that is easier to detect. That's what I would start with actually. Looks like you've got your job cut out for you, so good luck and don't despair - most of the things we do in the lab don't work. Life of a scientist. Makes the success so much sweeter!
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