I have hit a strange roadblock with my experiments. I am doing a ChIP experiment with an HA-tagged protein in H9c2 cells. I used IgG as an isotype control for my antibody, and after isolating DNA from IgG and HA did a qPCR with 1:100 input DNA. For my gene, regulator of calcineurin, I get a dilution-adjust Ct of 25 in my input sample, an average 30 Ct for my ChIP sample, and for the triplicate run of my IgG, I got 35.1, N/A, N/A. The melt curves looked great for the run. I'm not sure if I can quantify the IgG if I only got one signal. I would appreciate any advice on how I might want to interpret these results. I'm going to try to adjust the protocol to get more sensitivity and lower Ct's across the board, but as a preliminary result, I'd just like to see if I can do anything with the information I have.
Dear Ryan, if you want, I can take a look to your results and protocol.
My email is: [email protected]
By the way, take a look to this page where two methods for ChIP analysis are explained: https://www.thermofisher.com/es/en/home/life-science/epigenetics-noncoding-rna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html
As the IgG control is basically meant to measure your background, I suppose you should be happy to have almost no signal there. Perhaps just use the values you have from your HA samples to show what you pulled down (so without subtracting your IgG background). Also, once you repeat your experiment, you'll probably see if the IgGs are as good in every run, and then decide further how to normalize?
Using more DNA/reaction could indeed also help - to as you say get lower Ct values, and then perhaps your IgG will give you usable signal as well - might be good at least to try to verify if there's indeed no background, or if there's just too little input/reaction to detect...
Good luck!
Dear Ryan,
You could also consider trying to make a PCR on your immunoprecipitated DNA with your ab of interest, using primers that amplify a DNA region potentially not bound by your molecule of interest.
Best
Gilles
Dear Ryan:
How did u solve the problem?
I've done a similar experiment as yours, I used HA tag in HepG2 cells, also IgG as a negative control of my antibody. But my IgG CT value seems to be as high as my antibody's. Could you share with me your protocol? I'm wondering why I've got such a high noise signal!
Thanks a lot!
- Badges
- Science topic