Chip it kit

Manufactured by Active Motif

Sourced in United States

The ChIP-IT kit is a laboratory equipment product designed for chromatin immunoprecipitation (ChIP) assays. The kit provides the necessary components and protocols to perform ChIP experiments, which are used to identify DNA sequences that are associated with specific proteins of interest within a cell.

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Lab products found in correlation

Sc 11350x, by Santa Cruz Biotechnology (2 mentions) Control polyclonal non specific igg, by Cell Signaling Technology (2 mentions) Anti h3k27me3, by Merck Group (2 mentions) Syber green mix, by Bio-Rad (1 mentions) Ab42126, by Abcam (1 mentions) Prl cmv renilla plasmid, by Promega (1 mentions) Sc 3739, by Santa Cruz Biotechnology (1 mentions) Hiseq 2000, by Illumina (1 mentions) Runx2 m70 antibody, by Santa Cruz Biotechnology (1 mentions) Anti h3k4me3, by Merck Group (1 mentions) Anti rna polymerase 2, by Santa Cruz Biotechnology (1 mentions) Anti v5, by Thermo Fisher Scientific (1 mentions) Anti flag, by Thermo Fisher Scientific (1 mentions) Gapdh, by Active Motif (1 mentions) Rna pol 2, by Active Motif (1 mentions) Normal mouse igg, by Thermo Fisher Scientific (1 mentions) Sc 133074, by Santa Cruz Biotechnology (1 mentions) Ab4729, by Abcam (1 mentions) Model 100, by Thermo Fisher Scientific (1 mentions) Protein g dynal beads, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

ChIP-IT Expression Chromatin Immunoprecipitation Kits ChIP-ITTM kit ChIP-ITTM Express kit Re-ChIP-IT magnetic chromatin reimmunoprecipitation kit ChiP-IT Express Enzymatic Magnetic Chromatin Immunoprecipitation Kit Re-ChIP-IT kit ChIP-IT Express Magnetic Chromatin Immunoprecipitation kit Re-ChIP-IT sequential ChIP kit ChIP-IT Express kit ChIP-IT Express Enzyme kit

59 protocols using chip it kit

Chromatin Immunoprecipitation Assay

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Chromatin was isolated from 2 million cells according to the manufacturer’s recommended procedure in CHIP-IT kit (Active Motif 53040). Sheared chromatin (250 μg) was immunoprecipitated using 5 μg of ChIP quality antibody. ChIP-DNA was eluted (200 μl of elution buffer for ChIP-PCR or in 50 μl for ChIP-seq), and 2 μl were analysed by q-PCR using SYBER Green mix (Bio-Rad). Sheared chromatin (25 μg) were used as Input-DNA. Primers were designed by Prime3 and validated in the Genome Browser. Primers are described in Supplementary Methods.

Benitez J.A., Ma J., D’Antonio M., Boyer A., Camargo M.F., Zanca C., Kelly S., Khodadadi-Jamayran A., Jameson N.M., Andersen M., Miletic H., Saberi S., Frazer K.A., Cavenee W.K, & Furnari F.B. (2017). PTEN regulates glioblastoma oncogenesis through chromatin-associated complexes of DAXX and histone H3.3. Nature Communications, 8, 15223.

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FOXO1 Chromatin Immunoprecipitation in ATDC5 Cells

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Chromatin immunoprecipitation (ChIP) assays were performed with the ChIP-IT Kit (Active Motif, Carlsbad, CA) using approximately 1.5 x107 ATDC5 cells. The cells were cultured using multiple conditions, including 1) hypertrophic differentiation for 6 days using differentiation media as mentioned earlier; 2) cells at 70–80% treated with CML-BSA (200 mg/mL), an AGE, for 3 days or unmodified BSA (200 mg/mL) for a similar period; and 3) cells grown in High glucose (HG) (25 mmol/L) media for 5 days. Formaldehyde was used to fix the cells and nuclei obtained following Dounce homogenization. ChIP was performed following the manufacturer’s instructions using an anti-FOXO1 antibody (5 mg) (SC-11350X; Santa Cruz Biotechnology) or control polyclonal non-specific IgG (Cell Signaling Technology). Protein G-coupled beads were used to purify the chromatin–antibody complexes. Three quantitative real-time PCR reactions for the caspase-3 promoter region, which contains FOXO1 consensus response elements, were done with similar results. Experiments were performed with triplicate replicates and carried out three times with similar results.

Alharbi M.A, & Graves D.T. (2023). FOXO 1 deletion in chondrocytes rescues diabetes-impaired fracture healing by restoring angiogenesis and reducing apoptosis. Frontiers in Endocrinology, 14, 1136117.

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ChIP-seq analysis of ORAI3 promoter

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ChIP assays were performed on five million CD4⁺ T cells by using the ChIP-IT Kit (53040) from Active Motif. Oligonucleotide primers were designed to amplify the ORAI3 promoter sequence (chr16/30,961,071–137; forward: 5ʹ-TTGCTGTTATTCTGTGGTGAG-3ʹ and reverse: 5ʹ-CAAAATAAGGGATCCATCAGA-3ʹ). Normal IgG was used as control.

Ye Z., Shen Y., Jin K., Qiu J., Hu B., Jadhav R.R., Sheth K., Weyand C.M, & Goronzy J.J. (2021). Arachidonic acid-regulated calcium signaling in T cells from patients with rheumatoid arthritis promotes synovial inflammation. Nature Communications, 12, 907.

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Quantifying E2F3 Binding on PGC-1α

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ChIP assay was performed using the chip-IT kit (Active Motif), following the manufacturer’s instructions and as described elsewhere (81 (link)). E2F3 binding on the PGC-1α promoter was calculated by subtracting the intensity value of the band detected upon immunoprecipitation with the anti-E2F3 antibody from the intensity value detected from the input band, and the difference is presented as percentage arbitrary units.

Bahn Y.J., Yadav H., Piaggi P., Abel B.S., Gavrilova O., Springer D.A., Papazoglou I., Zerfas P.M., Skarulis M.C., McPherron A.C, & Rane S.G. (2023). CDK4-E2F3 signals enhance oxidative skeletal muscle fiber numbers and function to affect myogenesis and metabolism. The Journal of Clinical Investigation, 133(13), e162479.

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Regulation of DNA mismatch repair genes by NRIP1

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The MSH2 and MSH6 luciferase reporter and NRIP1 expression vectors were previously described [26 (link),27 (link),28 (link),29 (link)]. HCT116 cells were transfected in 96-well plates (2.5 × 10⁴ cells per well) 24 h prior to DNA transfection with Jet-PEI (275 ng of total DNA). Increasing doses of pEF-c-myc-RIP140 or pEF-c-myc-RIP^MSI were cotransfected with the pGL3-MSH2-Luc or the Sp1 mutant pGL3-MSH2m1-Luc (kind gifts of E. Huang [28 (link)]). Similar experiments were performed with the pGL3-MSH6-Luc reporter vector and a Sp1 mutant pGL3-MSH6M1-2/7-Luc (kind gifts of R.D. Kolodner [29 (link)]). The pRL-CMV-renilla plasmid (Promega, Charbonnières-les-Bains, France) was used to normalize transfection efficiency. Firefly luciferase values were measured and normalized by the Renilla luciferase activity. Values were expressed as the mean ratio of luciferase activities.
ChIP assays for proteins at the MSH2 and MSH6 promoters were performed in HT29 cells using the CHIP-IT kit (Active Motif, Carlsbad, CA, USA). Sonicated chromatin was immunoprecipitated with antibodies against IgG (sc-3739, Santa Cruz Biotechnology, Inc, Heidelberg, Germany), H3pan (CC16310135, Diagenode, Liège, Belgium) and NRIP1 (ab42126, Abcam, Paris, France). Immunoprecipitated DNA was amplified by qPCR using the primers listed in Supplementary Table S1.

Palassin P., Lapierre M., Pyrdziak S., Wagner A., Stehle R., Corsini C., Duffour J., Bonnet S., Boulahtouf A., Rodriguez C., Ho-Pun-Cheung A., Lopez-Crapez E., Boissière-Michot F., Bibeau F., Thezenas S., Elarouci N., Selves J., Hoffmann J.S., Roepman P., Mazard T., Buhard O., Duval A., Jalaguier S., Cavaillès V, & Castet-Nicolas A. (2021). A Truncated NRIP1 Mutant Amplifies Microsatellite Instability of Colorectal Cancer by Regulating MSH2/MSH6 Expression, and Is a Prognostic Marker of Stage III Tumors. Cancers, 13(17), 4449.

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ChIP-Seq Protocol for Transcription Factor Binding

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ChIP experiments were performed using ChIP-IT kit (Active Motif) as previously described^{71 (link)}. 5 µg of antibodies (Supplementary Table 5) were used. Immunoprecipitated-chromatin was eluted after several washes, reverse cross-linked and stored at −20 °C. ChIP-qPCRs were performed in the same way as RT-qPCR with 2 µL of ChIP or IgG samples instead of cDNA. Primer sequences are in Supplementary Table 5. Enriched DNA from EGR1-ChIP and input DNA fragments were used to generate libraries as previously described^{71 (link)}. Fifty-cycle single-end sequencings were performed using HiSeq 2000 (Illumina). Reads were aligned using human genome hg19 with BWA (v0.7.5a), peak calling assessed using MACS 2.0, and annotation done with HOMER (v4.7.2), with a p value of 0.01. Integrative Genomics Viewer (IGV 2.1) was used for representation.

Pronier E., Imanci A., Selimoglu-Buet D., Badaoui B., Itzykson R., Roger T., Jego C., Naimo A., Francillette M., Breckler M., Wagner-Ballon O., Figueroa M.E., Aglave M., Gautheret D., Porteu F., Bernard O.A., Vainchenker W., Delhommeau F., Solary E, & Droin N.M. (2022). Macrophage migration inhibitory factor is overproduced through EGR1 in TET2low resting monocytes. Communications Biology, 5, 110.

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Foxo1 Regulation of Inflammatory Genes

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Chromatin immunoprecipitation (ChIP) assays were performed using ChIP-IT Kit (Active Motif, Carlsbad, CA) following the manufacturer’s instructions. To precipitate Foxo1, anti-Foxo1 antibody was used, and the quantitative real-time PCR of CCL20 and IL-36γ promoters was performed.

Xu F., Othman B., Lim J., Batres A., Ponugoti B., Zhang C., Yi L., Liu J., Tian C., Hameedaldeen A., Alsadun S., Tarapore R, & Graves D.T. (2014). Foxo1 Inhibits Diabetic Mucosal Wound Healing but Enhances Healing of Normoglycemic Wounds. Diabetes, 64(1), 243-256.

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ChIP-Seq of Pol II, MEF2 and H3 in Jurkat Cells

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Jurkat cells growing exponentially were cross-linked in situ and subjected to nuclear isolation, DNA shearing, preclearing, and immunoprecipitation. Procedures used were modified from the ChIP-IT kit (Active Motif) using enzymatic DNA shearing as described previously in detail (Ramachandran, 2008 (link), Rathore et al., 2012 (link)). The Pol II (8WG16 monoclonal) ChIP antibody was from Covance. The MEF2 antibody was from Santa Cruz Technologies and the histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated) from Cell Signaling Technology.

Khan A.U., Rathore M.G., Allende-Vega N., Vo D.N., Belkhala S., Orecchioni S., Talarico G., Bertolini F., Cartron G., Lecellier C.H, & Villalba M. (2015). Human Leukemic Cells performing Oxidative Phosphorylation (OXPHOS) Generate an Antioxidant Response Independently of Reactive Oxygen species (ROS) Production. EBioMedicine, 3, 43-53.

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FOXO1 Regulation of RANKL in Chondrocytes

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Chromatin immunoprecipitation (ChIP) assays were carried out with the ChIP-IT Kit (Active Motif, Carlsbad, CA) using ∼1.5 × 10⁷ ATDC5 murine chondrocytes. The ATDC5 were cultured using different conditions, which included: 1) hypertrophic differentiation for 6 days using a 1:1 mixture of DMEM and Ham’s F12 medium supplemented with 5% FBS, 1% Anti-Anti, 50 µg/mL ascorbic acid, and 0.5 mmol/L NaH₂PO₄, 2) cells at 70–80% treated with CML-BSA (200 μg/mL) for 3 days or unmodified BSA (200 μg/mL) for 3 days, and 3) cells grown in HG (25 mmol/L) media for 5 days or mannitol (25 mmol/L) as osmotic control. Cells were fixed with formaldehyde and nuclei obtained following Dounce homogenization. ChIP was performed following the manufacturer’s instructions using an anti-FOXO1 antibody (5 μg) (SC-11350X; Santa Cruz Biotechnology) or control polyclonal nonspecific IgG (Cell Signaling Technology). Chromatin–antibody complexes were purified using protein G–coupled beads. Three quantitative real-time PCR reactions for the RANKL gene promoter with primers used were: forward, 5′-TGAAGACACACTACCTGACTCCTG-3′ and reverse, 5′-CCCACAATGTGTTGCAGTTC-3′, which flanks a consensus FOXO1 response element. The experiment was repeated three times with similar results.

Alharbi M.A., Zhang C., Lu C., Milovanova T.N., Yi L., Ryu J.D., Jiao H., Dong G., O’Connor J.P, & Graves D.T. (2018). FOXO1 Deletion Reverses the Effect of Diabetic-Induced Impaired Fracture Healing. Diabetes, 67(12), 2682-2694.

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Chromatin Immunoprecipitation of Runx2 in Chondrocytes

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Chromatin immunoprecipitation from chondrogenic cells was performed using ChIP-IT kit (Active Motif, Carlsbad, CA). Briefly, cells were cross linked in 1% formaldehyde/PBS for 10 minutes at 22°C and washed first with PBS and then glycine solution. Cells were collected in PBS containing 5mM PMSF, resuspended in lysis buffer, and sonicated 4 times for 20 seconds each. Gel electrophoresis confirmed that the bulk of DNA fragments from soluble chromatin were ~ 400–500 bp in length. Immunoprecipitation was carried out for 14 hours at 4°C with 5μg of Runx2 M70 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG as a negative control. The immunoprecipitated chromatin was collected using Protein A/G beads. Chromatin and 5% input were analyzed by real time PCR using primers designed to amplify promoter fragments spanning the RUNX motif in Sfn, Gpr132, Cyclin A1, and c-Myb genes. The primer sequences and location of RUNX binding sites in each gene are listed in Table S3. In parallel, conventional PCR was performed with 31~37 cycles of amplification. Amplicons were run on a 2% agarose gel, stained with ethidium bromide, and visualized under UV light. The cellular distribution of Runx2 protein and its interaction of Runx2 protein with target DNA were assessed by immunofluorescence and EMSA as described in supplement methods.

Chen H., Ghori-Javed F.Y., Rashid H., Adhami M.D., Serra R., Gutierrez S.E, & Javed A. (2014). Runx2 Regulates Endochondral Ossification through Control of Chondrocyte Proliferation and Differentiation. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 29(12), 2653-2665.

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