Small intestine tissues were washed with formaldehyde (37°C, 1% final concentration) and then opened longitudinally on a glass plate. The mucosa was scraped and separated from the underlying muscle layers by a glass microscope slide. The scraped mucosal cells were then fixed in formaldehyde (37°C, 1% final concentration) for 10 min at room temperature. The chromatin immunoprecipitation (ChIP) assay was performed as described previously [28 (link), 29 (link)] using the EpiQuik Tissue Chromatin Immunoprecipitation kit (P-2003, EpiGentek, Farmingdale, NY). Chromatin was immunoprecipitated with control IgG or anti-ChREBP antibody (Novus Biologicals), purified, and then analyzed by qPCR using a CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). The following primers were used: ChoRE1 forward 5′GATTTC CTG CCG CAT TCA GA3′, reverse 5′TTTTCAGAC CTC CCA GAT GGA3′; ChoRE2 forward 5′TCC ATC CAC ACA CTT TCA AAC C3′, reverse 5′CAA GCC ACG GCC AAC AG3′; ChoRE3 forward 5′TCC CCG GCT CAC CTC AA3′, reverse 5′TTC GGA GTG GGA GTC TGG TT3′; ChoRE4/5 forward 5′TGG TCA GTC CGG TAG CAG TTG3′, reverse 5′CCT TTG CAG GGC AGG CTA A3′; mNHE3 ChoRE1 forward 5′GGG AGG ATA TAG GGA ATT TG3′, reverse 5′CGA TAC TTG AAA CGT ATA TAT GT3′; mCyclophilin forward 5′GGT CTT TGG GAA GGT GAA AGA A3′, reverse 5′GCC ATT CCT GGA CCC AAA A3′.
Epiquik tissue chromatin immunoprecipitation kit
Manufactured by Epigentek
Sourced in United States
The EpiQuik Tissue Chromatin Immunoprecipitation (ChIP) kit is a laboratory tool designed to isolate and analyze protein-bound DNA fragments from tissue samples. The kit provides reagents and protocols to facilitate the chromatin immunoprecipitation process, which is a widely used technique in epigenetic research.
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Lab products found in correlation
Cfx384 touch real time pcr detection system, by Bio-Rad (2 mentions)
Sonicator, by Emerson (2 mentions)
Homogenizing buffer, by Epigentek (2 mentions)
Nuclear lysis buffer, by Epigentek (2 mentions)
Anti smp30 sc377184, by Santa Cruz Biotechnology (2 mentions)
Antibody diluted buffer, by Emerson (2 mentions)
Anti chrebp antibody, by Novus Biologicals (1 mentions)
Nebnext ultra 2, by New England Biolabs (1 mentions)
Dmem 5 mm glucose, by Thermo Fisher Scientific (1 mentions)
Tru seq kit, by New England Biolabs (1 mentions)
Nextseq instrument, by Illumina (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Normal mouse igg, by Epigentek (1 mentions)
Anti h3k4me3, by Epigentek (1 mentions)
Anti h3k27me3, by Merck Group (1 mentions)
S2 sonicator, by Covaris (1 mentions)
Ab8898, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
Iq sybr green, by Bio-Rad (1 mentions)
10 protocols using epiquik tissue chromatin immunoprecipitation kit
1
Chromatin Immunoprecipitation of ChREBP in Intestinal Mucosa
2
EIF3F ChIP-seq on A549 cells
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EIF3F-A549 and CTL-A549 cancer cells were grown in DMEM 5 mM glucose (Gibco), supplemented with 10% FBS (Gibco) and penicillin–streptomycin 1 × (100×; Gibco) for 24 h. We used 2.0 × 106 cells for classical cell lysis with a RIPA buffer supplemented with proteases and phophatases inhibitors. The EpiQuik Tissue Chromatin Immunoprecipitation Kit provided by Epigentek was performed following the manufacturer’s recommandations guidelines. For plate coating, 3 µg of antibodies have been used: EIF3F (Novus NBP2-16299) and EIF3F (Abcam ab155475). DNA libraries were prepared using Tru seq Kit and NEB Next ultra II following New England Biolabs (manual E7645) protocol. DNA fragments of 280 pb were quality analyzed with DNA high sensitivity chip on Bioanalyzer 2100. DNA sequencing was performed by Fasteris (https://www.fasteris.com/dna/ ) on Illumina Next seq instrument with 2 × 75pb reads. Fastq sequences were aligned and analyzed mapped on GRCh38 human genome using CLC software.
3
Transcription Factor Binding Site Prediction and ChIP Analysis
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Binding sites for transcription factors were predicted using the matrix60 (link), and the Regulatory Sequence Analysis Tools portal (http://rsat.ulb.ac.be/rsat/ , last accessed March 15, 2015). Testis samples (20 mg) from dmrt1bY:GFP transgenic fish and GFP antibody (Upstate) were used for in vivo chromatin immunoprecipitation (ChIP) by using the EpiQuik Tissue Chromatin Immunoprecipitation kit (Epigentek)16 (link)61 (link). After tissue disaggregation and cell re-suspension, DNA was sheared by sonication (9 pulses of 10s with an amplitude of 10%)16 (link). ChIP procedure and analysis of DNA enrichment by real-time PCR were as described16 (link)62 (link).
4
Chromatin Immunoprecipitation of ER-α and Histone Modifications
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Chromatin immunoprecipitation (ChIP) assays were performed using EpiQuik Tissue Chromatin Immunoprecipitation Kit (Epigentek) according to the instructions supplied with minor modification per manufacturer’s suggestions. Around 50 mg of tissue was cut into 1–2 mm3 pieces and cross-linked with 1% formaldehyde for 15 min at room temperature on a rocking platform. Subsequent cell lysis and DNA shearing were completed before incubation of tissue extracts with ERα (F-10X, Santa Cruz), anti-histone H4 acetyl K12 (ab46983, Abcam) or normal mouse IgG (Epigentek). After protein/DNA immunoprecipitation, reversal of cross-linked DNA was performed, and DNA was purified using a kit-supplied reagent (Epigentek). Primer for GREB1 [21 (link)] qRT-PCR: Forward-5′-GCCAAATGGAAGAAGGACAG-3′; Reverse-5′-ACCACCTACCTCCAGTCACC-3′ were used. Primer for TFF1 [39 (link), 40 (link)]: Forward-5′-GGCAGGCTCTGTTTGCTTAAAGAGCG-3′; Reverse-5′-GGCCATCTCTCACTATGAATCACTTCTGC-3′.
5
Chromatin Immunoprecipitation Profiling
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Hippocampal tissue samples were thawed on ice then treated with 1% formaldehyde for five minutes and sonicated with the truChIPTM Tissue Prep Kit for SDS Chromatin Shearing (Covaris) and the Covaris® S2 Sonicator (Woburn, MA, USA) according to the manufacturer’s protocol. The EpiQuik™ Tissue Chromatin Immunoprecipitation Kit (Epigentek) was used to perform ChIP. After sonication, samples were divided and immunoprecipitated with ChIP-grade polyclonal antibodies anti-H3K4me3 (Epigentek cat # A-4033) and anti-H3K27me3 (Millipore cat #07–499). Two microarray experiments were performed, one for each methylation state using the same chromatin sample from the same mice for each. Immunoprecipitated samples were sent to ArrayStar Inc. (Rockville, MD, USA). ArrayStar performed whole-genome amplification, target preparation DNA labelling and array hybridization.
6
Chromatin Immunoprecipitation of Ageing Mouse Brain
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Chromatin immunoprecipitation in brain of C57 mice at post-natal day 10 (PND 10) and PND 365 was performed using EpiQuik Tissue Chromatin Immunoprecipitation kit (Epigentek #P-2003) according to manufacturer’s instructions. Briefly, 150 mg of frozen tissue were cut into small pieces (<1 mm3) and cross-linked with 1% formaldehyde for 10 min at room temperature and then quenched in PBS 1X-Glycine 1.25 M for 10 min at room temperature. The samples were homogenized using a Douncer homogenizer and centrifuged to pellet nuclei. After homogenization, lysis buffer was added to nuclei. Chromatin was prepared and sonicated using a water bath Bioruptor (Diagenode; 30” ON/30” OFF, High power, 3 × 10 cycles) to a size range of 200–1000 bp. To pre-cleared cell debris, sonicated chromatin was centrifuged at 12,000 x g at +4 °C for 10 min. Chromatin was diluted and ChIP performed according to manufacturer’s instructions using antibodies against H3K9me3 (ab8898, Abcam), histone H3 (ab1791, Abcam), IgG1 (G3A1, Cell Signalling) was used as negative control in the immunoprecipitation. Immunoprecipitated DNA was purified by phenol-chloroform extraction and in parallel 5 ul (5%) were taken to be used as input in the quantification analysis. qPCRs were performed using iQ SYBR Green in a CFX96 Real-Time PCR system (Bio-Rad). Primer sequences are reported in Table S1 .
7
Chromatin Immunoprecipitation Assay Protocol
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ChIP-qPCR assays of MEEC line and mice cardiac tissues were performed using the chromatin immunoprecipitation assay kit (Millipore) and EpiQuik Tissue Chromatin Immunoprecipitation Kit (Epigentek) following the protocol supplied by the manufacturer. Primers used for ChIP-qPCR are listed in Supplementary Table S5 .
8
SMP30 Chromatin Immunoprecipitation in Milk Sheep Liver
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Chromatin immunoprecipitation (ChIP) was performed using an EpiQuik™ Tissue Chromatin Immunoprecipitation Kit (Epigentek, Farmingdale, NY, USA). 40 mg milk sh livers were cross-linked with 1% formaldehyde, and unreacted formaldehyde was quenched by 125 mM glycine. The liver was then homogenized in homogenizing buffer (Epigentek) using a plastic rod in the 1.5 mL microcentrifuge tube with 10-20 strokes. After centrifugation (5000 g, 5 min, 4°C), the pellets were suspended in the nuclear lysis buffer (Epigentek) and washed by phosphate-buffered saline (PBS). The cell lysis was conducted with 5 pulses of 20 sec using a sonicator (Branson, Brook eld, CT, USA), and the resulting DNA was found to be between 200-1,000 bp. The recoating-strip was incubated with a polyclonal antibody against anti-SMP30 (sc377184, Santa Cruz) or IgG (Epigentek) as the negative control at room temperature for 90 min. The coated-strip was washed twice with the antibody-diluted buffer (Branson). The protein-DNA complexes were reverse crosslinked using Proteinase K incubated at 65°C for 15 min. The extracted DNA was puri ed using spin columns for cleanup. For qPCR analysis, immunoprecipitated DNA and input DNA (un-immunoprecipitated) were used as templates. The qPCR primers for ChIP-qPCR are listed in Table S1. Subsequent qPCR analysis followed the previously explained methods.
9
SMP30 Chromatin Immunoprecipitation in Milk Sheep Liver
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Chromatin immunoprecipitation (ChIP) was performed using an EpiQuik™ Tissue Chromatin Immunoprecipitation Kit (Epigentek, Farmingdale, NY, USA). 40 mg milk sh livers were cross-linked with 1% formaldehyde, and unreacted formaldehyde was quenched by 125 mM glycine. The liver was then homogenized in homogenizing buffer (Epigentek) using a plastic rod in the 1.5 mL microcentrifuge tube with 10-20 strokes. After centrifugation (5000 g, 5 min, 4°C), the pellets were suspended in the nuclear lysis buffer (Epigentek) and washed by phosphate-buffered saline (PBS). The cell lysis was conducted with 5 pulses of 20 sec using a sonicator (Branson, Brook eld, CT, USA), and the resulting DNA was found to be between 200-1,000 bp. The recoating-strip was incubated with a polyclonal antibody against anti-SMP30 (sc377184, Santa Cruz) or IgG (Epigentek) as the negative control at room temperature for 90 min. The coated-strip was washed twice with the antibody-diluted buffer (Branson). The protein-DNA complexes were reverse crosslinked using Proteinase K incubated at 65°C for 15 min. The extracted DNA was puri ed using spin columns for cleanup. For qPCR analysis, immunoprecipitated DNA and input DNA (un-immunoprecipitated) were used as templates. The qPCR primers for ChIP-qPCR are listed in Table S1. Subsequent qPCR analysis followed the previously explained methods.
10
ChIP Assay for Transcription Factors
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The chromatin immunoprecipitation (ChIP) assay was performed as described previously (Jeong et al. 2011 ) using the EpiQuik Tissue Chromatin Immunoprecipitation kit (P-2002, EpiGentek, NY, USA). Briefly, Nthy-ori 3-1 (3 × 10 6 cells), KTC-2 (3 × 10 6 cells), BCPAP (3 × 10 6 cells) and MDA-T32 (3 × 10 6 cells) cells were cross-linked with 1% formaldehyde and blocked with 1.25 M glycine solution. The cells were resuspended in CP3 lysis buffer containing a protease-inhibitor cocktail, and sonicated until the cross-linked chromatin was sheared to an average DNA fragment length of 200-300 bp. Chromatin was immunoprecipitated with control IgG (#2729; Cell Signaling technology), anti-ETV1 (sc-55581; Santa Cruz Biotechnology), anti-ETV4 (ARP32263_P050, Aviva Systems Biology, CA, USA) and anti-ETV5 (13011-1-AP, Proteintech, IL, USA), purified, and then analyzed by qPCR using a CFX384 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). KTC-2, BCPAP and MDA-T32 cells were treated with 1 μM of PLX4720 for 24 h. The primers were validated by analysis of template titration and dissociation curves. The primer sequences are listed in Supplementary Table 2.
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