ChIP Anti-bodies HDAC1

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.

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Found 3 matching solutions for this experiment

Protocol tips
The sheared cross-linked chromatin was immunoprecipitated (IP) using ChIP-validated antibodies to acetyl-histone H3, acetyl-histone H3- Lys9 (H3K9), trimethyl-histone H3-Lys9 (Upstate Biotechnology), HDAC1 and RNA Pol II (Santa Cruz Biotechnology). Each IP reaction consisted of 125 μl of chromatin + 375 μl of dilution buffer with protease inhibitors + 20 μl of protein G magnetic beads + 5 μg of primary antibody. The IP reactions were incubated at 4° C overnight with rotation. IgG from the same species as the primary antibodies served as negative controls. Magnetic beads were separated using a magnetic separator (Biolabs) and the supernatant was discarded. The Protein G magnetic beads-antibody-chromatin complex was incubated with a series of wash buffers provided in the Magna ChIP G tissue Kit: one time each for 5 min each wash on a rotating platform followed by magnetic clearance and careful removal of the supernatant fractions: 500 μl low salt immune complex wash buffer, 500 μl high salt immune complex wash buffer, 500 μl LiCl immune complex wash buffer, 500 μl TE buffer.
HDAC1 (D5C6U) XP® Rabbit mAb #34589

Cell Signaling Technology

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 10 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 2 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.
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