ChIP Mouse - Ovary

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

Chromatin Immunoprecipitation (ChIP) Assay 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

EZ-Magna ChIP™ G - Chromatin Immunoprecipitation Kit

Merck Millipore

Upstream tips	Protocol tips	Downstream tips
- DO NOT cryo-preserve specimen with sucrose. -Make fresh 1% formaldehyde before each experiment.	-Keep lysate ice-cold. Sonication produces heat, which can denature the chromatin. Allow time between cycles of sonication to prevent sample overheating. - DNA fragments should be in 200 -1000 bps in size. -Add 1-10 µg of immunoprecipitating antibody.

Upstream tips
- DO NOT cryo-preserve specimen with sucrose. -Make fresh 1% formaldehyde before each experiment.
Protocol tips
-Keep lysate ice-cold. Sonication produces heat, which can denature the chromatin. Allow time between cycles of sonication to prevent sample overheating. - DNA fragments should be in 200 -1000 bps in size. -Add 1-10 µg of immunoprecipitating antibody.

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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