Product Reviews: MED7 antibody – C-terminal region (ARP38446_P050) in HCT116 using CHIP

The product page for Aviva’s MED7 antibody – C-terminal region (ARP38446_P050) can be found here.

Quiescent human colon carcinoma HCT116 cultures were treated with 10% FBS for three time points (0, 15, 30min) or (0, 30, 60min) were used in Matrix-ChIP and real-time PCR assays at EGR1 gene (Exon1) and 15kb upstream site.

 

 

Protocol

Matrix-ChIP utilizes sheared chromatin. Capture antibodies are surface-immobilized via blocked, Protein A coated 96-well plates. Blocking buffer consists of 5% BSA and 100ug/mL sheared salmon sperm DNA in immunoprecipitation (IP) buffer. After washing, sheared chromatin samples are added and the plates are floated in an ultrasonic water bath (Bronson 8510) to accelerate protein-antibody binding. Wells are then washed and DNA eluted with Tris base (pH 9.8) and stored (4C) in the same Matrix ChIP plates for repeated use. Plates are sealed with adhesive film to prevent evaporation and can be stored/re-sealed for months for repeated qPCR.

Real-time PCR is performed with 2X SYBR Green PCR master mix, eluted DNA template and primers in a 2-4uL final volume in a 384-well Optical Reaction Plate. Amplification (three steps, 40 cycles), data acquisition and analysis were run in qudriplicate for each PCR reaction.

-Amount of chromatin input equivalent to 200-500ng DNA (100uL at 2-5ng/uL)
-Amount of antibody used: 0.5ug
-Fold molar excess of blocking peptide: 100
-Antibody:Blocking peptide incubation time: 30min at RT
-Antibody:chromatin incubation time: 60min at 4C in ultrasonic water bath (Branson 8510)
-ChIP assays done in duplicate
-All time points are in minutes

Microplate-based chromatin immunoprecipitation method, Matrix-ChIP: a platform to study signaling of complex genomic events.
Flanagin S, Nelson JD, Castner DG, Denisenko O, Bomsztyk K.
Nucleic Acids Res. 2008 Feb;36(3)

Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays.
Yu J, Feng Q, Ruan Y, Komers R, Kiviat N, Bomsztyk K.
BMC Mol Biol. 2011 Nov 18;12:49.

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