ChIP Mouse - Podocyte

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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Found 3 matching solutions for this experiment

Pierce™ Agarose ChIP Kit

Thermo Fisher Scientific

Upstream tips
-If you are unfamiliar with cell type being used, culture an extra dish of cells for determining cell number.
Before crosslinking, trypsinize and determine the cell number from the extra dish of cells.
Protocol tips
-After incubation, it is essential to open the column cap before plug removal to equalize the pressure within the column. Failure to open the cap before plug removal will result in sample loss.
Downstream tips
-If you are preparing chromatin in bulk, unused supernatant may be stored at -80°C for later use.
Upstream tips
-Do not use formaldehyde if white precipitate is visible in the solution.
Protocol tips
-Use ChIP validated antibody.
- 1X104 cells per ChIP when using high affinity antibodies directed towards more abundant proteins.
-Protease Inhibitor Cocktail III contains DMSO and will remain frozen below 18.4°C.
-Keep cell lysate ice cold. Sonication produces heat, which can denature the chromatin. Allow at least 30
seconds between cycles of sonication to prevent sample overheating.
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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