Chip it high sensitivity

Manufactured by Active Motif

Sourced in United States

The ChIP-IT High Sensitivity is a laboratory equipment product that facilitates chromatin immunoprecipitation (ChIP) experiments. It is designed to provide high sensitivity for the detection and analysis of protein-DNA interactions.

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Lab products found in correlation

Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions) Chip buffer, by Active Motif (1 mentions) D2k6w, by Cell Signaling Technology (1 mentions) Light cycler 480 sybr green 1 master x2 kit, by Roche (1 mentions) Ab60134, by Abcam (1 mentions) Sybr green system, by Bio-Rad (1 mentions) Adi spa 896 f, by Enzo Life Sciences (1 mentions) Ab 105 c, by R&D Systems (1 mentions)

Spelling variants of this product

ChIP-IT High Sensitivity kit (168 citations) ChIP-IT Express Enzymatic and ChIP-IT High Sensitivity kit (2 citations)

4 protocols using chip it high sensitivity

Chromatin Immunoprecipitation of VDR

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Mouse duodenum was harvested and washed twice in ice-cold PBS, incubated in PBS containing 1% paraformaldehyde (PFA, Electron Microscopy Sciences) for 10 min, and quenched with 0.125 M glycine at room temperature for 5 min. Nuclei from cross-linked tissues were isolated in ChIP Buffer (ChIP-IT High Sensitivity, Active Motif) following the manufacturer’s procedure and sonicated in 300–400 bp DNA fragments. Thirty µg of chromatin was immunoprecipitated with anti-VDR antibodies (D2K6W, Cell Signaling) and DNA was recovered following the manufacturer’s directions (ChIP-IT High Sensitivity, Active Motif). qPCR reactions were performed using the Light Cycler 480 SYBR Green I Master X2 Kit (Roche) according to the supplier’s protocol. Oligonucleotides are listed in Supplementary Table 3.

Rovito D., Belorusova A.Y., Chalhoub S., Rerra A.I., Guiot E., Molin A., Linglart A., Rochel N., Laverny G, & Metzger D. (2020). Cytosolic sequestration of the vitamin D receptor as a therapeutic option for vitamin D-induced hypercalcemia. Nature Communications, 11, 6249.

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ChIP-qPCR Assay for Transcription Factors

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We performed ChIP by using cross-linked lysate from FLNC homozygous KO iPSC-CMs and control iPSC-CMs according to the manufacturer’s instructions (ChIP-IT High Sensitivity; ACTIVE MOTIF; catalog no. 53040). iPSC-CMs were fixed with formaldehyde to cross-link the protein/DNA complexes. DNA was then sheared into small fragments by sonication; the sheared DNA was then incubated with anti–β-catenin (ChIP grade) and precipitated using Protein G agarose beads to pull down the complexes of interest. Next, the pull-down cross-linked products were reversed and digested with Proteinase K, and the DNA was recovered and purified for qPCR. The primers for SOX2 binding region were 5′-TTCAGACCACCCAGTCTTGT-3′ and 5′-CCGTTTTGAACTAGGGGTCT-3′; primers for SOX9 binding region were 5′-AGGCCTTTAAACCTTCCAGA-3′ and 5′-AAAGCAGAAAGCAATGGATG-3′; NFKB primers were 5′-ATCAGAGAGCGATGAAGGTG-3′ and 5′-GATGCTCCAGGAACCAGAC-3′.

Chen S.N., Lam C.K., Wan Y.W., Gao S., Malak O.A., Zhao S.R., Lombardi R., Ambardekar A.V., Bristow M.R., Cleveland J., Gigli M., Sinagra G., Graw S., Taylor M.R., Wu J.C, & Mestroni L. (2022). Activation of PDGFRA signaling contributes to filamin C–related arrhythmogenic cardiomyopathy. Science Advances, 8(8), eabk0052.

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ChIP Assay for Pathogenic Th17 Cells

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ChIP assay was performed according to the manufacture’s protocol of ChIP-IT High Sensitivity (Active Motif). Briefly, 7×10⁶in vitro differentiated pathogenic Th17 cells were cross-linked with 1% formaldehyde, then chromatin was sheared by sonication for 25 min (cycle of 30 sec on, 30 sec off). Chromatin was incubated with 4 μg of anti-RORα (ab60134, Abcam) or control rabbit IgG overnight at 4 °C and then incubated with pre-washed protein G agarose beads for 3 hrs. ChIP reactions were spun 1250 × g for 1 min, washed three times and immunoprecipitated DNA was purified. Il17a CNSs were analyzed by RT-PCR with SYBR green system (Bio-Rad) using primers for Il17a CNS1 and Il17a CNS2 listed in the Key Resource Table.

Shimada K., Porritt R.A., Markman J.L., Gire O’rourke J., Wakita D., Noval Rivas M., Ogawa C., Kozhaya L., Martins G.A., Unutmaz D., Baloh R.H., Crother T.R., Chen S, & Arditi M. (2018). T cell intrinsic Receptor Interacting Protein 2 regulates pathogenic T helper-17 cell differentiation. Immunity, 49(5), 873-885.e7.

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ChIP-seq Analysis of Hmox1 in iPSCs

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ChIP analysis was carried out according to the protocol ChIP-IT High Sensitivity (Active Motif, Carlsbad, CA, USA). Mouse Hmox1^+/+ and Hmox2^+/+ iPSCs were seeded in six-well plates and were cultured until they were approximately 90% confluent. To increase the Hmox1 levels, cells were stimulated for 4 h with 2 µmol/L hemin. An input step was made to check the correctness of the chromatin isolation (adding NaCl and heating at 100 °C steps were omitted). Immunoprecipitation was performed using an anti-Hmox1 antibody (#ADI-SPA-896-F, Enzo Life Sciences, Villeurbanne, France) and control rabbit IgG (#AB-105-C, R&D Systems, Minneapolis, MN, USA). Six micrograms of antibodies were used per sample. The final step was performed to check the potential DNA sequences bound by HO1. For the detection of potential DNA sequences bound to Hmox1 protein, an electrophoretic separation was performed on a 1% agarose gel with ethidium bromide.

Krzeptowski W., Chudy P., Sokołowski G., Żukowska M., Kusienicka A., Seretny A., Kalita A., Czmoczek A., Gubała J., Baran S., Klóska D., Jeż M., Stępniewski J., Szade K., Szade A., Grochot-Przęczek A., Józkowicz A, & Nowak W.N. (2021). Proximity Ligation Assay Detection of Protein–DNA Interactions—Is There a Link between Heme Oxygenase-1 and G-quadruplexes?. Antioxidants, 10(1), 94.

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