I already checked in Raw 264.7 macrophages cell line .But i didn't find the Nf-kb p65 phosphorylation band .if anyone has suggestion how can i get the Nf-kb p65 phosphorylation band ?
1.Should i reduce my induction concentration ? or
2.Should i reduce my induction time ?if i reduce my induction time how long time will be suitable to get a phosphorylation band.
Did you use the adequate phosphatase inhibitors when you harvest the sample protein?
Our work group use a [ ] of LPS 1 ug/mL but changed dependently of lot. and specie used to obtain the LPS, after stimuli of macrophagues for 1 hour, how many time u incubate your samples (cells)? because the complete activation its around 6 to 24 hours dependly to conditions
Yes i use adequate phosphatase inhibitor at a concentration of 1:10 with RIPA buffer.
Hi Rahima,
In my experience, NF-kB should be activated in macrophages by 30 min. What is your treatment condition? Did the cells were at least 70% confluent before treatment? Did you use serum free medium for treatment? Did you extract cytosolic and nuclear fractions separately? Before going for nuclear fraction, I would suggest to try with whole cell fraction. If still you don't get the signal, I would like to ask you the passage number of your cells. I don't go for more than 30. If you are using older passage, then try with a new vial.
With best
thanks for your kindly information. I treat the cell with 5 ug/mL LPS for 1 hour.Now i reduce my induction time from 1 hour to 30 min.
I had about 50-60% cell confluency. Yes i used serum free medium and the whole cell fraction for getting protein. Usually i used 18-20 passage number of cells.Should i try to reduce my induction concentration to get the signaling.
My treatment condition is molecular hydrogen which has big role as an antioxidant and anti inflammatory.
Hi Rahima,
I think the confluency of your cells also could be an issue. Cells usually like to be in contact with each other and therefore, you could try with >70% confluent cells. I also use highest 1 ug/ml of LPS. You also try with this rather than 5 ug/ml.
With best
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