ChIP Human - Kupffer Cells

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 2 matching solutions for this experiment

EZ-Magna ChIP™ G - Chromatin Immunoprecipitation Kit

Merck Millipore

Upstream tips	Protocol tips	Downstream 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.

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.

Manufacturer protocol Publication protocol 1 Relevant paper

OneDay ChIP kit

Diagenode

Upstream tips	Protocol tips	Downstream tips
	-Each ChIP requires sheared chromatin from 1.5 to 2x 10e6 cells. - If you work with yeast rather than with cultured cell lines, you might need to block the beads with BSA. -Do not disturb the pellet. The supernatant contains the formed [antibody-chromatin] complexes. -Do not spin the beads at high speed.

Protocol tips
-Each ChIP requires sheared chromatin from 1.5 to 2x 10e6 cells. - If you work with yeast rather than with cultured cell lines, you might need to block the beads with BSA. -Do not disturb the pellet. The supernatant contains the formed [antibody-chromatin] complexes. -Do not spin the beads at high speed.

Publication protocol 1 Relevant paper

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