Hey,

I'm using normal rabbit IgG as negative control for my experiments (since I have rabbit antibodies). I had to optimize the protocol for not getting so much unspecific binding, but now I'm successful to get a wuite low negative control and medium to high target results. 

Of course you can also use only beads as control to show that there's no unspecific binding of your protein to the beads. But I suggest to use also the control with antibody, since you have to show that your results are specific for your antibody of interest!

Hi Oki,

If the IgG and TF Ab have similar Cts, around the~mid-20’s, then it may be that more stringent or additional washes are needed. If the IgG and TF Ab have higher similar Cts, Cts>30’s, then antibody may not be ChIP-grade, if input chromatin is enough.

As control, you can use beads alone, but they may also give a high background; and the IgG may be a better control, as it will account for non-specific binding with both, beads and Ab. You can add a pre-clearing step with only agarose beads if desired, and this may help reduce the background, also you may switch to using magnetic beads instead.

You can also design primers to a genomic region in your particular cells/tissues where the TF of interest does not bind and use this as an additional background/negative control.

Good luck!

Blanca

Dear Blanca L. Valle

Thank you for the answer.

Hi. I checked PCR analysis. I saw melting curve shifting in PCR.

I tried gel electrophoresis. I saw several bands. 

I made another primer in a similar region. But I got the same result. However, I saw the different intensity in product size.

I think primers form dimer or hairpin. But the sequence what I want is so hard to pick up primer.  

My point is what if I use only product size band in several bands in PCR analysis by gel electrophoresis. 

regards.

Dear Ariane Eberhardt 

Thank you for the answer.

Hi. I tried chip assay again via reducing non-specific product. 

I figure out that the problem is primers. It might form dimer or hairpin. 

Could you give me some tips how did you design primer specific region?

I tried design specific region, it has so lots of G/C contents that it forms dimer or hairpin.

Regards.

What is the reason for getting the same CT values in beads only sample and the

desired antibody sample?