ChIP Human - Fibroblast cell lines

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™ Magnetic ChIP Kit

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream 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.	-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.	-If you are preparing chromatin in bulk, unused supernatant may be stored at -80°C for later use.

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.

Manufacturer protocol Publication protocol 1 Relevant paper

EZ-Magna ChIP™ A/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

ChIP-IT® Express Chromatin Immunoprecipitation Kits

Active Motif

Upstream tips	Protocol tips	Downstream tips
-Do not re-freeze the Protein G magnetic beads, the beads should be stored at 4C.	-Always use ChIP validated antibody. -Optimal use of 1-3 ug of antibody.	-The diluted Proteinase K stop solution can not be stored.

Upstream tips
-Do not re-freeze the Protein G magnetic beads, the beads should be stored at 4C.
Protocol tips
-Always use ChIP validated antibody. -Optimal use of 1-3 ug of antibody.
Downstream tips
-The diluted Proteinase K stop solution can not be stored.

Manufacturer protocol Publication protocol 1 Relevant paper

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