ChIP Human - HUVEC

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

EZ-ChIP™

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

SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
-Once in solution, store 1M DTT at -20°C.	-For optimal ChIP results, use approximately 4 X 106 cells for each immunoprecipitation to be performed. -For optimal ChIP results, it is highly critical that the chromatin is of appropriate size and concentration. -For optimal ChIP results, use approximately 5 to 10 µg of digested, cross-linked chromatin per immunoprecipitation.

Upstream tips
-Once in solution, store 1M DTT at -20°C.
Protocol tips
-For optimal ChIP results, use approximately 4 X 106 cells for each immunoprecipitation to be performed. -For optimal ChIP results, it is highly critical that the chromatin is of appropriate size and concentration. -For optimal ChIP results, use approximately 5 to 10 µg of digested, cross-linked chromatin per immunoprecipitation.

Manufacturer protocol Publication protocol 1 Relevant paper

TruSeq ChIP Library Preparation Kit

Illumina

Upstream tips	Protocol tips	Downstream tips
-Remove End Repair Mix and Resuspension Buffer from -15°C to -25°C storage and thaw them at room temperature.	-5–10 ng ChIP-enriched, fragmented input DNA is recommended. -Carefully collect ChIP DNA samples to make sure that they are free of contaminants.

Upstream tips
-Remove End Repair Mix and Resuspension Buffer from -15°C to -25°C storage and thaw them at room temperature.
Protocol tips
-5–10 ng ChIP-enriched, fragmented input DNA is recommended. -Carefully collect ChIP DNA samples to make sure that they are free of contaminants.

Manufacturer protocol Publication protocol 1 Relevant paper

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