EZ-ChIP™

Protocol tips

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.

Publication protocol

ING1 binding to the promoter of miR-371-5p was tested using ChIP analysis. ChIP assay was performed using the EZ-ChIP kit from Millipore (Millipore, USA) according to the manufacturer's instructions. Briefly, about 3×106 Panc-1, infected with either Ad-GFP-ING1 or Ad-GFP adenoviruses were crosslinked using 1% formaldehyde (BIO-RAD, USA) for 15 min at 37°C. Cells were harvested after quenching with 0.125 M glycine and lysed in ChIP lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris (pH 8.0), 0.1% deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 µg/ml pepstatin, 1 µg/ml aprotinin and 1 µg/ml leupeptin). Extracts were sonicated six times for 9 s each and lysates were spinned by centrifugation at 14000 rpm for 20 min at 4°C. Of this sample, 150 µl was used as input. The supernatants were immunoprecipiated with either anti-ING1 or with mouse IgG (negative control) at 4°C for 4 h, followed by protein G Sepharose (Sigma, USA) for 2 h at 4°C. The immunoprecipitates were sequentially washed with 1 ml of ChIP lysis buffer twice, ChIP lysis buffer with 500 mM NaCl twice and with LiCl/detergent solution (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA) twice and finally with TE buffer (10 mM Tris and 1 mM EDTA, pH 8.0). The beads were eluted using 1% SDS and 0.1 M sodium bicarbonate solution. The input and the eluent samples were reverse-crosslinked using NaCl for 5 h at 65°C. The DNA from the samples was isolated by phenol–chloroform, followed by ethanol precipitation. Promoter binding was tested by PCR using primers spanning the upstream regions of miR-371-5p start sites, primer sequences: ChIP-ING1, F: 5′-AATGTTCACTGCCGCACTGT-3′; R: 5′-ACACCTATAATCCCTGC- TAC-3′; ChIP-neg-F: 5′- ACGGCCAACATGCTCAGG-3′; R: 5′- TGTTTGCAACTGCTGCGTTAG-3′.

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 Human - PANC-1 using EZ-ChIP™ from Merck Millipore.

Manufacturer protocol

Download the product protocol from Merck Millipore for EZ-ChIP™ below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing ChIP Human - PANC-1 using EZ-ChIP™ from Merck Millipore. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.