Pierce™ Magnetic ChIP Kit

Protocol tips

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.

Publication protocol

Chromatin immunoprecipitation (ChIP) assays were performed as previously described (14,19). Cross-linked 832/13 or NIH 3T3-L1 chromatin (1 mg) was incubated overnight (18 h) at 4°C with 3 μg rabbit anti-ChREBP (ab157153; Abcam) antibody, anti-Myc antibody (N-262, sc-764; Santa Cruz Biotechnology), or normal rabbit IgG (Santa Cruz Biotechnology). Immune complexes were captured with 60 μL 50% protein A-Sepharose agarose slurry (Millipore #16-157). DNA-protein crosslinking was reversed by adding 50% Chelex beads and boiling 10 min. Quantitative PCR using SYBR Green was performed in 5 µL supernatant containing immunoprecipitated DNA fragments, as previously described (14,19).

For ChIP from frozen liver tissue, liver tissue was finely powdered and fixed with 1% formaldehyde. The powder was homogenized with a Dounce homogenizer with tight (A) pestle in a hypotonic solution (10 mmol/L HEPES [pH 7.9], 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.2% Nonidet P-40, 0.2 mmol/L sodium orthovanadate, 0.15 mmol/L spermine, 0.5 mmol/L spermidine, 1 mmol/L EDTA, 5% sucrose, 1 mmol/L dithiothreitol, and protease inhibitors) and layered onto a sucrose cushion buffer (10 mmol/L Tris/HCl [pH 7.5], 15 mmol/L NaCl, 60 mmol/L KCl, 0.15 mmol/L spermine, 0.5 mmol/L spermidine, 1 mmol/L EDTA, 10% sucrose, and protease inhibitors). Sucrose gradient centrifugation was performed for 1 h at 30,000 rpm. The pellet was washed once with ice-cold PBS and resuspended in SDS lysis buffer. The sample was then sonicated with a Bioruptor and processed as described above.

For rat islets, we used a micro-ChIP assay. Briefly, rat islets cultured with 20 mmol/L glucose culture medium for 2 days were crosslinked with 1% formaldehyde, followed by neutralization with 125 mmol/L glycine. After fixation, a ChIP assay was performed using the Thermo Scientific Pierce Magnetic ChIP Kit (Cat. #26157), according to the manufacturers’ instructions. Antibodies to ChREBP (Thermo Scientific, Cat. #PA5-22924) and IgG control antibody (Santa Cruz Biotechnology, Cat. #2027) were used to immunoprecipitate chromatin-bound supernatants.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing ChIP Rat - Pancreas using Pierce™ Magnetic ChIP Kit from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Pierce™ Magnetic ChIP Kit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing ChIP Rat - Pancreas using Pierce™ Magnetic ChIP Kit from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.