Chip it express

Manufactured by Active Motif

Sourced in United States

ChIP-IT Express is a kit designed for chromatin immunoprecipitation (ChIP) experiments. It provides the necessary reagents and protocols to perform ChIP assays efficiently.

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Lab products found in correlation

Chip it express enzymatic shearing kit, by Active Motif (3 mentions) Chromatin ip dna purification kit, by Active Motif (3 mentions) Sybr green master mix, by Thermo Fisher Scientific (3 mentions) Anti flag antibody, by Merck Group (2 mentions) Anti igg antibody, by Merck Group (2 mentions) Normal rabbit igg, by Cell Signaling Technology (2 mentions) Ab5131, by Abcam (2 mentions) Stepone real time pcr system, by Thermo Fisher Scientific (2 mentions) Anti h3.3 17 10245, by Merck Group (2 mentions) Dynabead, by Thermo Fisher Scientific (2 mentions) Vector nti advance 10, by Thermo Fisher Scientific (1 mentions) Ab8895, by Abcam (1 mentions) H3k27me3, by Active Motif (1 mentions) Monoclonal anti flag, by Merck Group (1 mentions) Histone h3, by Active Motif (1 mentions) Bioruptor ucd 250, by Cosmo Bio (1 mentions) Qia pcr purification kit, by Qiagen (1 mentions) Chip dna clean concentrator, by Zymo Research (1 mentions) Faststart universal sybr green master, by Roche (1 mentions) Sybr green, by Thermo Fisher Scientific (1 mentions)

35 protocols using chip it express

Taurine Modulates TXNIP Expression

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Caco-2 cells were cultured in 6-well plates and incubated with taurine for 48 h. The cells were then homogenized, and the nuclear extract was prepared using the ChIP assay. The ChIP assay was performed using ChIP-IT Express (Active Motif, Carlsbad, CA, USA), according to the manufacturer’s instructions. To quantify the number of DNA fragments containing the TXNIP promoter region bound by Ets-1 protein, real-time PCR was performed. The primer sequences were as follows: human TXNIP promoter, 5′-TCGGATCTTTCTCCAGCAAT-3′ (forward), and 5′-AAATCGAGGAAACCCCTTTG-3′ (reverse).

Satsu H., Gondo Y., Shimanaka H., Imae M., Murakami S., Watari K., Wakabayashi S., Park S.J., Nakai K, & Shimizu M. (2022). Signaling Pathway of Taurine-Induced Upregulation of TXNIP. Metabolites, 12(7), 636.

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ChIP Assay for β1-Integrin Transcriptional Regulation

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The chromatin immunoprecipitation (ChIP) assay was performed using the ChIP-IT Express magnetic chromatin immunoprecipitation kit (Active Motif, Carlsbad, CA) according to the manufacturer’s protocol. Panc1, L3.6pL, MiaPaCa2, RKO and SW480 cancer cells were treated with DMSO, DIM-C-pPhOH (15, 20 μM), or DIM-C-pPhCO₂Me (15, 20 μM) for 24 hr. Cells were then fixed with 1% formaldehyde, and the cross-linking reaction was stopped by addition of 0.125 M glycine. After washing twice with phosphate-buffered saline, cells were scraped and pelleted. Collected cells were hypotonically lysed, and nuclei were collected. Nuclei were then sonicated to the desired chromatin length (~200 to 1,500 bp). The sonicated chromatin was immunoprecipitated with normal IgG, p300 (Santa Cruz), Sp1 (Abcam), NR4A1 (Novus Biologicals), Sp3, Sp4 (Santa Cruz), or RNA polymerase II (pol II; Active Motif) antibodies and protein A-conjugated magnetic beads at 4°C for overnight. After the magnetic beads were extensively washed, protein-DNA cross-links were reversed and eluted. DNA was prepared by proteinase K digestion followed by PCR amplification. The primers for detection of the β1-integrin promoter region were 5′-TCACCACCCTTCGTGACAC -3′ (sense) and 5′-GAGATCCTGCATCTCGGAAG-3′ (antisense). PCR products were resolved on a 2% agarose gel in the presence of RGB-4103 GelRed Nucleic Acid Stain.

Hedrick E., Lee S.O, & Safe S. (2017). The Nuclear Orphan Receptor NR4A1 Regulates β1-Integrin Expression in Pancreatic and Colon Cancer Cells and Can Be Targeted by NR4A1 Antagonists. Molecular carcinogenesis, 56(9), 2066-2075.

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Identifying p53 Repressive Response Elements in ENO3 Gene

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We used UCSC Genome Browser (http://genome.ucsc.edu/) to obtain the human ENO3 genomic sequence. The potential p53 repressive response elements (p53RREs), which are consisting of four canonical p53-binding sites (5’-RRRCW-3′) arranged head to tail and matching >90%, were identified by Vector NTI Advance 10 software (Invitrogen).
ChIP assay was performed with ChIP-IT Express (Active Motif) following the manufacturer's protocol. A-204 cells were fixed with 1% formaldehyde on a shaking platform for 10 min at room temperature following by sonication to shear the chromatin. Samples were immunoprecipitated with mouse monoclonal anti-p53 antibody or control mouse IgG serum (10 μg/ml) for PCR amplification. CD44 served as the positive p53RRE control [21 (link)].
Sequences of RT-PCR primer pairs for the putative p53RREs:
ENO3-RRE1 (Foward:5′-CAGGCAATGTCTGGATCACCG-3′, Reverse:5′-CTGACTGCGAAGAAACCCAAAG-3′)
ENO3-RRE2 (Foward:5′-CCTGTCTAAATTCGTTTCCTGTCC-3′, Reverse:5′-CACCCCAGGATTACATTCCC-3′)
ENO3-RRE3 (Foward:5′-TTGACCTTTGGTAAGGGGGC-3′, Reverse:5′-AGCCAATACCATGCTCACCC-3′)
CD44-RRE (Foward:5′-TTTACGGTTCGGTCATCCTC-3′, Reverse:5′-TGCTCTGCTGAGGCTGTAAA-3′)

Huang J., Du J., Lin W., Long Z., Zhang N., Huang X., Xie Y., Liu L, & Ma W. (2019). Regulation of lactate production through p53/β-enolase axis contributes to statin-associated muscle symptoms. EBioMedicine, 45, 251-260.

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Chromatin Immunoprecipitation Protocol

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ChIP assays were carried out using a kit (ACTIVE MOTIF, ChIP-IT Express, catalog #53008). Briefly, cells (2 × 10⁷) in a 10 cm culture dish were treated with 1% formaldehyde to cross-link chromatin-associated proteins to DNA. The cell lysates were subjected to ultrasound for 9–10 sets of 10-s pulses at 40% output to shear the DNA into fragments between 200 and 1000 bps. Equal cell lysates were respectively incubated with 1 μg of anti-Flag antibody (Sigma) and anti-IgG antibody (Millipore) as negative control. All the above chromatin supernatants were incubated with 20 μl magnetic proteinG beads overnight at 4 °C with rotation. Second day, the protein-DNA complexes were reversed and purified for pure DNA. The human SNAI1 promoter was amplified with RT-PCR.

Jiao H.L., Weng B.S., Yan S.S., Lin Z.M., Wang S.Y., Chen X.P., Liang G.H., Li X.Q., Zhao W.Y., Huang J.Y., Zhang D., Zhang L.J., Han F.Y., Li S.N., Chen L.J., Zhu J.H., He W.F., Ding Y.Q, & Ye Y.P. (2020). RETRACTED ARTICLE: Upregulation of OSBPL3 by HIF1A promotes colorectal cancer progression through activation of RAS signaling pathway. Cell Death & Disease, 11(7), 571.

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ChIP Assay for MFG-E8 Promoter

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The assay was performed using ChIP-IT^™ Express (Active Motif, CA). In brief, 0.54 ml of 37% formaldehyde was added to RAW 264.7 cells in a 15-cm cell culture dish containing 20 ml of culture medium. Cells were incubated on a shaking platform for 10 min at room temperature for cross-linking of DNA to proteins. Treated cells were then washed with Glycine Stop-Fix Solution and followed by fragmentation of chromatin DNA with sonication and immunoprecipitation of targeted protein-chromatin DNA complexes with an antibody against Sp1 or c-Jun using a protocol for ChIP assay provided by the manufacturer. Then, the isolated protein-chromatin DNA complexes were heated at 65°C for 2.5 h for reverse crosslinking, followed by deproteinization with proteinase K. Finally, MFG-E8 promoter bound to Sp1 or c-Jun were detected using PCR with the following sets of primers: Sp1-F 5′-AATTTCCCCCTGTCCAGTCTAT-3′ and Sp1-R 5′-CTCCTCTCACTCCGGAATAAATC-3′ for the −87 to +31 region; AP-1F 5′-AGGAAACAGGGTCCCATTCT-3′ and AP-1R 5′-AGCATGCTGGAGACCCTAGA-3′ for the −466 to −266 region. The non-specific primers (NSP) used for the post-ChIP PCR were NSP-F 5′-TCCCAAGTGCTGGGATTAAGG-3′ and NSP-R: 5′-AACTCTCAGCTCCTCCTGCACC -3′. The NSP primers detected a region located at about 2.5 kb from −466 nt upstream of the MFG-E8 promoter. The PCR products were analyzed with 2% agarose gel.

Wang X., Bu H.F., Liu S.X., De Plaen I.G, & Tan X.D. (2015). Molecular mechanisms underlying the regulation of the MFG-E8 gene promoter activity in physiological and inflammatory conditions. Journal of cellular biochemistry, 116(9), 1867-1879.

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Histone Modification ChIP Assay

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We prepared sonicated DNA samples from AGS and MKN7 cells using a ChIP-IT Express (No. 39163, Active Motif), and then incubated with 1-3 μg of three Histone H3 methylation-related (H3K4me3, No. 39915; H3K9me3, No. 29766; and H3K27me3, No. 39155; Active Motif) and two histone acetylation-related antibodies (acetyl-Histone H3 (K9 and K14), #06-599, acetyl-histone H4 (K5, K8, K12, K16), #06-866; Upstate). Moreover, after transfection of the SET7/9 expression vector or its siRNA into GC cells, ChIP assays were conducted using anti-H3K4me1 polyclonal (ab8895, Abcam, Cambridge, UK) and anti-FLAG monoclonal (Sigma-Aldrich Japan) antibodies. Histone H3 (No. 39163, Active Motif) and Normal Rabbit IgG (No. 2729, Cell Signaling Technology) were used as positive and negative controls, respectively. Input DNA samples were used as internal controls. The primer sequences and their conditions are shown in Supplementary Table S2.

Akiyama Y., Koda Y., Byeon S.J., Shimada S., Nishikawaji T., Sakamoto A., Chen Y., Kojima K., Kawano T., Eishi Y., Deng D., Kim W.H., Zhu W.G., Yuasa Y, & Tanaka S. (2015). Reduced expression of SET7/9, a histone mono-methyltransferase, is associated with gastric cancer progression. Oncotarget, 7(4), 3966-3983.

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ChIP-seq Analysis of H3K27ac in SLFN11-KO Cells

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Twenty-five million CCRF-CEM parental and SLFN11-KO
cells were treated or untreated with CPT (100 nM) for 4 hours. ChIP assay
was done by following the instruction manual of ChIP-IT Express (Active
Motif). Briefly, cells were fixed with 1% formaldehyde in medium for 10 min
at room temperature. Fixation was stopped with x1 glycine/PBS. Cells were
lysed with lysis buffer with proteinase inhibitor cocktail and PMSF and
homogenized 60 times by small tight homogenizer. Cell pellets were
re-suspended with sharing buffer, and sonicated 30 minutes by the following
settings: 30 s on, 30 s off, level at H, (Bioruptor XL). Five %/volume of
each sample was saved as input. The left of the supernatant was incubated
with 2 μg antibody (H3K27ac or normal rabbit IgG) and protein G beads
for overnight at 4°C. After reversing cross-link and proteinase K
treatment, immunoprecipitated DNA was purified with ChIP DNA Clean &
concentrator-5 (ZYMO Research) according to the manual. DNA libraries were
constructed following standard protocol and submitted to the Center for
Cancer Research Sequencing Facility (Frederick).

Murai J., Zhang H., Pongor L., Tang S.W., Jo U., Moribe F., Ma Y., Tomita M, & Pommier Y. (2020). Chromatin Remodeling and Immediate Early Gene Activation by SLFN11 in Response to Replication Stress. Cell reports, 30(12), 4137-4151.e6.

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Chromatin Immunoprecipitation Protocol

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ChIP was performed using ChIP-IT express (Active Motif) as previously described (Birdsey et al., 2012 (link)).

Birdsey G.M., Shah A.V., Dufton N., Reynolds L.E., Osuna Almagro L., Yang Y., Aspalter I.M., Khan S.T., Mason J.C., Dejana E., Göttgens B., Hodivala-Dilke K., Gerhardt H., Adams R.H, & Randi A.M. (2015). The Endothelial Transcription Factor ERG Promotes Vascular Stability and Growth through Wnt/β-Catenin Signaling. Developmental Cell, 32(1), 82-96.

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ChIP-IT Express Quantitative Assay

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We used ChIP-IT Express (Active Motif, Carlsbad, CA) to perform ChIP assays. Briefly, chromatin complexes isolated from cells fixed with 1% formaldehyde were sonicated (Bioruptor UCD-250, Cosmobio, Tokyo, Japan) and immunoprecipitated by control IgG or RNA polymerase II antibody. The IL-8 promoter region contained in the immunoprecipitated samples were quantitated on a real-time PCR machine. The primers and probe set for IL-8 promoter region was as follows; Forward primer, 5′-CATCAGTTGCAAATCGTGGA-3′, Reverse primer, 5′-AGAACTTATGCACCCTCATCTTTT-3′, probe, Roche Universal ProbeLibrary Probe #6 (Roche applied Science).

Tomita T., Ieguchi K., Coin F., Kato Y., Kikuchi H., Oshima Y., Kurata S, & Maru Y. (2014). ZFC3H1, a Zinc Finger Protein, Modulates IL-8 Transcription by Binding with Celastramycin A, a Potential Immune Suppressor. PLoS ONE, 9(9), e108957.

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ChIP-qPCR Analysis of Cyp2b10 Regulation

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After sacrifice by cervical dislocation, liver samples were collected and ChIP samples were prepared as reported previously [24] (link). ChIP assays were performed using ChIP-IT Express (Active Motif, Carlsbad, CA) according to the manufacturer's protocol with 10 µg of sheared chromatin and 2 µg of anti-human RXRα antibody (Santa Cruz, D-20X), anti-RNA polymerase II phospho Ser-5, an activated form (ab5131, Abcam, Cambridge, UK), anti-histone H3 lysine 27 trimethylation antibody (H3K27me3; 39155, Active Motif) or control rabbit IgG (2729, Cell Signaling, Danvers, MA). After purification of ChIP DNA samples with QIA PCR purification kit (QIAGEN), Cyp2b10 enhancer region containing PBREM (−2440/−2238) and promoter region containing TATA box (-155/+69) were amplified by PCR using the following specific primers: 5′-GCTAATGCCTGTCTGGATCAGGA-3′ and 5′-GGAATACTGACCCAAGTTCAGTG-3′ (PBREM); 5′-AAGGGAATGAGGAGTGAGC-3′ and 5′-CAAGAAGCCCACAAGGAGAG-3′ (TATA). PCR reactions were performed with Green Taq PCR polymerase (Promega) at 94°C for 2 min followed by 30-35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s.

Ohno M., Kanayama T., Moore R., Ray M, & Negishi M. (2014). The Roles of Co-Chaperone CCRP/DNAJC7 in Cyp2b10 Gene Activation and Steatosis Development in Mouse Livers. PLoS ONE, 9(12), e115663.

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