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!

Hi Liu Chengyang,

I am also facing similar problem like yours and not able to solve it. Could you share your protocol with me for solving the problem?

Thanks a lot