Since January, I'm not able to see any phosphorylation of both AKT, STAT3 and STAT5. Even when I try western blotting or phospho flow cytometry.
I'm sure that there is no problem with,
- antibodies
- protocols
- me (because I gave my cells to other person and she found the same result as me)
Could it be related to the cell culture?
To be sure that it is not a problem of your antibodies you should:
- in case you are using a phosphoprotein specific antibody (i.e. an anti-pSTAT5) verify that this same antibody detectes the phosphoprotein in another cell line
- in case you are using a generic anti-phosphoresidue antibody verify that it can detect other phosphorylated proteins, except the three you have cited
Actually, very often it is a problem of antibodies...
Other possible pitfalls could concern:
- the phosphatases inhibitor you add to your lysis buffer
- the conditions in which you perform your experiment: if you were looking at post-stimulus phosphorylations, it could be that your stimulus does not work properly so that time of stimulation, concentration etc should be reconsidered (usually such phosphorylation events are transient)
If you have had the cells in culture for some time, it could be the result of DNA methylation of whatever genes are involved in triggering the pSTAT5. Treating with azacytidine might restore the expression.
I have a few tips for you:
1. Sample preparation. In addition to phosphatase inhibitor, proteinase inhibitor should also be added into your lysis buffer. After stimulation for a certain time, you have to put your cell plates on ice right away, and use ice-cold reagents for your sample preparation.
2. Use a new vial of frozen cells to set up a new culture. Make sure the cell density is proper, say about 80%, when you treat the cells.
3. Increase the loading amount of cell lysate.
4. Increase primary antibody concentration and incubation time.
5. Increase exposure time when you develop the film.
Good luck!
Dear all,
I did all what you suggest to me for WB, but there is no changes !!
1- Sample preparation : befor lysis, harvest cells at 10^6 cells (max), add phophatase and protease inhibitors in lysis buffer (pH7.5)
2- For WB :
- Increase the loading amount on cell lysate = 20 ng, 40 ng, 50 ng, 70 ng
- Primary antibody are from cell signaling, so the concentration is 1:1000 and the incubation is done at 4°C overnight
- When I increase exposure time, I obtained an ugly film
However I didn't try to treat cells with azacytidine
Regarding phosphoflow, I still not understand what happen, because before, my cells express clearly phospho STAT5 and phospho AKT and since one year, they express a very low signal !!!
Please help me !
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