ChIP Human - SH-SY5Y

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
	107 cells were cultivated in DMEM with 10% FBS in the absence or presence of 10 nM leptin for 1 hour. Cells were washed in 1X PBS and incubated with 1% formaldehyde for 10 minutes. Cells were lysed and sonicated using a Branson digital sonifier with 30% impulse power for six impulses. For immunoprecipitation, precleared cell lysates were incubated with anti–P-Y705-STAT3 (9131; Cell Signaling Technology) or mouse IgG (Santa Cruz Biotechnology Inc.) as a negative control.

Protocol tips
107 cells were cultivated in DMEM with 10% FBS in the absence or presence of 10 nM leptin for 1 hour. Cells were washed in 1X PBS and incubated with 1% formaldehyde for 10 minutes. Cells were lysed and sonicated using a Branson digital sonifier with 30% impulse power for six impulses. For immunoprecipitation, precleared cell lysates were incubated with anti–P-Y705-STAT3 (9131; Cell Signaling Technology) or mouse IgG (Santa Cruz Biotechnology Inc.) as a negative control.

Protocol tips

107 cells were cultivated in DMEM with 10% FBS in the absence or presence of 10 nM leptin for 1 hour. Cells were washed in 1X PBS and incubated with 1% formaldehyde for 10 minutes. Cells were lysed and sonicated using a Branson digital sonifier with 30% impulse power for six impulses. For immunoprecipitation, precleared cell lysates were incubated with anti–P-Y705-STAT3 (9131; Cell Signaling Technology) or mouse IgG (Santa Cruz Biotechnology Inc.) as a negative control.

Manufacturer protocol Publication protocol 1 Relevant paper

SOLiD™ ChIP-Seq Kit, with ChIP magnet

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
-The 1.25 M glycine must be at room temperature before use.	- Use 10,000–300,000 cells or 0.167–5 mg of tissue. -Whenever possible, use an antibody that is qualified for ChIP. - Never mix the beads by vortexing; do not allow the beads dry out. -Keep the formaldehyde incubation time and method consistent between samples. -Keep the cell lysate cooled on ice during sonication.

Upstream tips
-The 1.25 M glycine must be at room temperature before use.
Protocol tips
- Use 10,000–300,000 cells or 0.167–5 mg of tissue. -Whenever possible, use an antibody that is qualified for ChIP. - Never mix the beads by vortexing; do not allow the beads dry out. -Keep the formaldehyde incubation time and method consistent between samples. -Keep the cell lysate cooled on ice during sonication.

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

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