ChIP Rat - Brain

DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.

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Upstream tips
-If any reagent has formed a precipitate, warm at 55 °C until dissolved. Allow to equilibrate to room temperature before use.
Protocol tips
-Use 1 µg of antibody per well.
-The range of cells that can be used with this kit is 0.2–2.5 x 105/well/ChIP sample. Using more than 0.5 million cells per well will increase the non-specific binding and reduce the yield and specificity of the ChIP reaction. If higher yield of ChIP DNA is desired for downstream applications (e.g. ChIP-Seq., ChIP-chip): a) DNA could be pooled by eluting consecutively (with 50 µl of elution buffer) from multiple individual ChIP reactions.
Upstream tips
-Warm IP Lysis Buffer to room temperature to prevent precipitation.
-Prepare one extra plate of cells to estimate cell number.
-Thaw Protease Inhibitor Cocktail (PIC) at room temperature.
-It is recommended to take some trial to optimize sonication condition before beginning with sonication.
Protocol tips
-In general it is recommended that one million mammalian cells are required for each IP
fraction.
-Centrifuge the sample at 6000 × g for 5 min at 4 °C to eliminate bubbles.
-To reduce background, remove as much supernatant as possible in the last wash. Continue with next step OR store at 4°C overnight.
Protocol tips
-Just because an antibody works well in a Western blot does not always indicate it will perform well in chromatin immunoprecipitation.
Unlike a Western blot that detects proteins that have been denatured, a ChIP antibody must recognize the target protein in its native state. Use ChIP-validated antibody.
-Use 2-10 μg of your ChIP antibody depending on the abundance of your protein target.
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