ChIP Anti-bodies ERα

In ChIP, the most vital step is the binding of an antibody and choosing the right antibody. The binding affinity of different types of immunoglobulins to protein A or G differs significantly. Henceforth, it is recommended to choose either protein A or protein G coated beads. If you do not see any product in the positive control, add 5–10 μg of chromatin and 1–5 μg of antibody to each IP reaction and incubate with antibody overnight and an additional 2 hr after adding Protein G/A beads. If no product is observed in the experimental sample, add more DNA to the PCR reaction or increase the number of amplification cycles. Furthermore, if you have any problem with antibodies, make sure to use the ChIP-validated antibody.

Start discussion

No discussions found

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Found 3 matching solutions for this experiment

Upstream tips
-It is highly critical that the chromatin is of appropriate size and concentration.
Protocol tips
-For optimal ChIP and ChIP-seq results, use 5 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP.
-It is important to keep the tissue cold to avoid protein degradation.
-Use fresh formaldehyde that is not past the manufacturer's expiration date.
Downstream tips
-Once in solution, store 1M DTT at -20°C.
Upstream tips
-Add protease inhibitors to all lysis solutions before use.
Protocol tips
-Use 4 µg for 25 µg of chromatin.
-Keep cells on ice between the rounds of homogenisations.
- Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line.
Protocol tips
Four µg of ERα antibody (AER304, mouse monoclonal, Millipore) or mouse non-immune IgG (Millipore) was utilized for IPs.
Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!
Become shareholder Discussions About us Contact Privacy Terms