ChIP assay was performed by using ChIP kits (9003, Cell Signaling Technology) according to the manufacturer’s instructions. The primers used for ChIP are listed in Supplemental Table 2 .
Chip kit
Manufactured by Cell Signaling Technology
Sourced in United States
The ChIP kit is a laboratory tool used to investigate protein-DNA interactions. It enables the isolation and analysis of DNA fragments that are bound to specific proteins of interest within the cell.
Automatically generated - may contain errors
Lab products found in correlation
Chip grade antibody against zeb1, by Merck Group (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Anti stat3 antibody, by Cell Signaling Technology (1 mentions)
Tb greentm premix extaqtm 2, by Roche (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Protein a agarose beads, by Merck Group (1 mentions)
Pias3, by Santa Cruz Biotechnology (1 mentions)
Tri methyl histone h3 lys27, by Cell Signaling Technology (1 mentions)
Tri methyl histone h3 lys4, by Cell Signaling Technology (1 mentions)
Acetyl histone h3 lys23, by Merck Group (1 mentions)
Smad2, by Cell Signaling Technology (1 mentions)
Smad3, by Cell Signaling Technology (1 mentions)
Stat3, by Santa Cruz Biotechnology (1 mentions)
Smad4, by Santa Cruz Biotechnology (1 mentions)
Cyclin a, by Merck Group (1 mentions)
Taqman primer, by Thermo Fisher Scientific (1 mentions)
Cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
β tubulin antibody, by Abcam (1 mentions)
Tgf β elisa kit, by R&D Systems (1 mentions)
Cyclin e, by Merck Group (1 mentions)
57 protocols using chip kit
1
Chromatin Immunoprecipitation (ChIP) Protocol
2
Investigating STAT3 Binding in Astrocytes
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We investigated STAT3 binding to target genes in astrocytes (4–5 × 107/L) using ChIP kits (Cell Signaling Technology, Danvers, MA, United States) as described by the manufacturer. Cells were immobilized in 1% formaldehyde, cultured under OGD or normal (5% CO2, 37°C) conditions, then incubated with protease and phosphatase inhibitors. Separated chromatin was homogenized using a tissue grinder to extract DNA fragments of 200–1,000 base pairs. The DNA fragments were then incubated with an anti-STAT3 antibody (Cell Signaling Technology Inc.) that binds to magnetic beads. After separation and elution from the beads, reverse crosslinking proceeded as described by the manufacturer. The DNA was analyzed by quantitative PCR using primers.
3
Chromatin Immunoprecipitation (ChIP) Assay
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The chromatin immunoprecipitation (ChIP) assay was performed using a ChIP kit (Cell Signaling Technology, Danvers, MA, USA). Cells were cultured to a density of 70%–80% (10-cm dish) collected after drug stimulation. Crosslinking was performed with 1% formaldehyde. After fixation, 10× glycine units were added to terminate the cross-linking process. After three times of washing with PBS, the sample was collected in a 1.5-mL centrifuge tube, and 1 µL of 100 mM PMSF and 1 µL of PIC were added. The sample was resuspended in 1 mL of ChIP-lysis buffer. Ultrasound treatment was performed 30 min after ice lysis to concentrate chromatin DNA fragments of 500–1000 bp. The sample was centrifuged at 12000 RPM at 4 °C for 10 min, and the supernatant was collected in a new centrifuge tube. After the DNA fragments were separated and purified, chromatin fragmentation was observed using 12% agarose gel electrophoresis. The number of immunoprecipitation (IP) and Ig G groups was calculated according to the concentration. Proteins were captured via immunoprecipitation using the corresponding primary antibody or control IgG and incubated overnight at 4°C. Further collection and purification of chromatin fragments was performed, followed by protein unlinking at 65°C. The precipitated DNA fragments were analyzed using PCR.
4
Chromatin Immunoprecipitation Assay Protocol
5
Chromatin Immunoprecipitation (ChIP) Protocol
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Approximately 4 × 106 cells were collected for each ChIP reaction. Cross-linked cells were collected according to the instructions, and ChIP lysate was added for ultrasonic treatment to fragment the chromatin. Then, 50 μL of the solution containing cross-linked chromatin fragments was purified using a ChIP kit (Cell Signaling Technology), and 10 μL of the resulting purified DNA was subjected to 1% agarose gel electrophoresis to observe the size of the DNA fragments. The cell lysates of different treatment groups were combined with YY1 (Proteintech) and IgG (negative control; Abcam) for the ChIP assay, and the bound DNA was purified for quantitative analysis by qPCR. TB GreenTM Premix ExtaQTM II was used to perform qRT–PCR analysis on a LightCycler 480 II instrument (Roche), and the ChIP-enrichment rate was calculated according to the experimental results.
6
ChIP Assay for STAT1 and c-Myc Promoters
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The primers used for ChIP assays were: 5'-CACGGAGGTCAGTTGCTAAA-3′ (forward) and 5'-AGAAGGACGTGCTGTGTTTG-3′ (reverse) for the STAT1 promoter; 5'-ACTCAGTCTGGGTGGAAGGTATC-3′ (forward) and 5'-AGATAGGGAGGAATGATAGAGGC-3′ (reverse) for the c-Myc promoter. The cells were prepared to perform ChIP assay with the ChIP Kit (Cell Signaling Technology) according to the manufacturer's instructions as described previously 17 (link), 18 (link).
7
CTCF Regulation in A549 Cells
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A549 cells were transfected with pcDNA-CTCF or CTCF small interfering RNA and cultured in an incubator containing 5% CO2 at 37°C for 24 h. After removal of the medium, the cells were fixed with 16% paraformaldehyde. They were divided into IgG+siNC group, CTCF+siNC group, IgG+siCTCF group and CTCF+siCTCF group. CTCF-bound DNA was captured using antibodies according to the ChIP kit (Cell signaling Technology, Boston, MA, USA) instructions. PCR was used to verify the capture of CTCF gene promoter DNA, and ChIP-PCR was used for quantitative analysis. Immunoprecipitation efficiency was calculated using input sample percentage method.
8
ChIP Assay of ATF3 Binding
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Chromatin immunoprecipitation (ChIP) assays were performed using a ChIP KIT according to the manufacturer’s instructions (Cell signaling, 9002, USA). Cultured MDA-MB-231 and MDA-MB-435 cells were treated with 1% formaldehyde. Cell lysates were then sonicated and immunoprecipitated using an anti-ATF3 antibody (Santa Cruz, sc-518032, USA), or normal rabbit IgG (MultiSciences, GAR0072, China) as a control. The primer sequences used for ChIP analysis were as follows: forward primer (5ʹ-GGGCTGGTGTTGCCATTTCT-3ʹ), reverse primer (5ʹ-TTGGGGCAGGGTCGGACCTA-3ʹ). Amplified DNA was normalized to the input total DNA.
9
Chromatin Immunoprecipitation Assay for Epigenetic Regulation
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The ChIP kit was purchased from Cell Signaling Technology (Boston, MA, USA), and the experiments were performed according to the manufacturer’s instructions. Equal volumes of chromatin were immunoprecipitated with anti-Sp1, anti-EZH2, anti-HDAC3, and anti-trimethyl-histone H3 Lys27 (H3K27me3) or normal IgG as a negative control. Then, spin columns were used to purify DNA, followed by qPCR. The primers used for ChIP assays are listed as follows: promoter region (-2,000~-1,500): sense 5′-CAACTCATGCCCTTGTGGG-3′, antisense 5′-CGTGGGAACTGTGGTGGC-3′; promoter region (-1,500~-1,000): sense 5′-GCCTCTAGTGACCTGACGG-3′, antisense 5′-GGGAGATTCCAGGGCTGA-3′; promoter region (-1,000~-500): sense 5′-AGCTCAGGCATCTCCACA-3′, antisense 5′-CACCAGGGTTGTAATAAGAAT-3′; promoter region (-500~0): sense 5′-AAAAGCACCCGCGACCAC-3′, antisense 5′-GCAGCAAAGAAGGGAGGA-3′.
10
Chromatin Immunoprecipitation of OCT4 and NCOR1
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Chromatin immunoprecipitation (ChIP) assays were fulfilled using anti-OCT4 or anti-NCOR1 antibody (Cell Signaling Technology) and protein A-agarose beads (Millipore) as described according to the ChIP kit (Cell Signaling Technology) protocols. The p53 promoter DNA in the ChIP product was measured by qPCR using gene-specific primers. The PCR primer sequences are 5′-GGGTGAGTGGGATGGAAG-3′ (forward) and 5′-CGGGTGGATGTGCAA AGA-3′ (reverse).
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