ChIP Mouse - Kidney

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

EpiQuik Chromatin Immunoprecipitation (ChIP) Kit

Epigentek

Upstream tips	Protocol tips	Downstream tips
-Ensure that all buffers are in clear solution. Shake or vortex if these buffers precipitate.	-When processing spin columns, always cap spin columns before placing them in the microcentrifuge. -The conditions of crosslinked DNA shearing can be optimized based on cells and sonicator equipment. If desired, remove 5 µl of sonicated cell lysate for agarose gel analysis. The length of sheared DNA should be between 200-1000 bp.

Upstream tips
-Ensure that all buffers are in clear solution. Shake or vortex if these buffers precipitate.
Protocol tips
-When processing spin columns, always cap spin columns before placing them in the microcentrifuge. -The conditions of crosslinked DNA shearing can be optimized based on cells and sonicator equipment. If desired, remove 5 µl of sonicated cell lysate for agarose gel analysis. The length of sheared DNA should be between 200-1000 bp.

Manufacturer protocol Publication protocol 1 Relevant paper

SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005

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

ChIP-IT High Sensitivity®

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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