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Beyochip chip assay kit

Manufactured by Beyotime
Sourced in China

The BeyoChIP™ ChIP Assay Kit is a tool used for chromatin immunoprecipitation (ChIP) experiments. ChIP is a technique used to study the interaction between proteins and DNA within a cell. The kit provides the necessary reagents and protocols to perform ChIP assays.

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6 protocols using beyochip chip assay kit

1

Chromatin Immunoprecipitation Assay for ZNF148

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ChIP assay was performed using BeyoCHIPTM CHIP Assay Kit (Beyotime) according to the manufacturer's instructions. T‐DCs were fixed via the addition of formaldehyde (final concentration of 1%) for 10 min, then administering glycine was to neutralize extra formaldehyde. Cells were washed with PBS, lysed with SDS Lysis Buffer for 10 min, then sonicated under 4°C to shear the cross‐linked DNA to 200–1000 bp. The cross‐linked protein‐DNA was immunoprecipitated with magnetic beads, and protein‐DNA complexes were precipitated using IgG and anti‐ZNF148 antibody (Abclonal) overnight. After that, the eluted DNA fragments were detected with PTX3 promoter‐specific primers and SYBR premix.
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2

RNA-seq and ChIP-PCR Analysis of YY1 in CRC

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Total RNA was isolated from control or siYY1 LOVO cells, and were sent for RNA-sequencing, which was performed in a Genergy Biotechnology company (Shanghai, China). Chromatin immunoprecipitation (ChIP) assay was performed using the BeyoChIPTM ChIP Assay Kit (Beyotime, China) in HCT116, LOVO, and DLD1 cells according to the manufacturer’s instructions. DNA was purified using a PCR purification kit (Tiangen, China) and prepared for PCR analysis. Then, agarose gel electrophoresis of the PCR product was carried out in 2% agarose gel containing DNA Gel dye. The primers used for ChIP-PCR analysis were listed in Supplementary Methods and Table S2.
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3

Chromatin Immunoprecipitation for DEC1

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ChIP assays were carried out according to the instructions of the BeyoChIP™ ChIP Assay Kit (Beyotime, Shanghai, China, P2080S). Briefly, the cells were subjected to fixation in 1.0% v/v formaldehyde for 15–20 min at room temperature, followed by quenching with 100 mM glycine. The cells were subsequently disrupted using a mini beadbeater, and the crosslinked chromatin was fragmented by sonication using a Bioruptor (7 cycles of 30 s on/off). The resulting extracts were then incubated for either 2 h or overnight at 4 ˚C with magnetic Prot A beads that were conjugated with a polyclonal antibody against DEC1 (Proteintech, Wuhan, China, 17895-1-AP). Sub-sequently, the reverse cross-linking process was conducted in Tris-EDTA buffer consisting of 100 mM Tris pH 8.0, 10 mM EDTA, and 1.0% v/v SDS at a temperature of 65 °C overnight. Following a proteinase K treatment of 2 h, the samples were subjected to a cleanup process, and the enrichment of DEC1 was determined through real-time PCR using SYBR green mix. The chromatin DNAs that were precipitated underwent Quantitative PCR using the identical procedure as that of qRT-PCR. The percentage of input DNA was utilized to determine the relative enrichment of the DNAs.
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4

Chromatin Immunoprecipitation Assay for VEGFA Promoter

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The ChIP assay was conducted using the BeyoChIP™ ChIP Assay Kit (Beyotime) in accordance with a previously documented methodology. In summary, the cells OE-NC and OE-HOOK3 MKN-28 were subjected to a 10-minute treatment with 37% formaldehyde in order to induce crosslinking of the chromatin. The chromatin was subjected to sonication using suitable ultrasonic parameters (performed on ice; at 10% power; with a 3-s sonication followed by a 10-s pause; repeated 16–18 times) in order to generate DNA fragments spanning a range of 100–1000 base pairs in length. The immunoprecipitation procedure was conducted utilizing a rabbit anti-SP1 antibody obtained from Proteintech. As a negative control, normal rabbit IgG was employed. Following the de-crosslinking of the complexes, the purified chromatin underwent PCR amplification with specialized primers that were created specifically for the VEGFA promoter region. The DNA fragments obtained were subsequently subjected to analysis using agarose gel electrophoresis. Please see Supplementary Table S1 for the primer sequences.
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5

ChIP Assay for Protein-DNA Interactions

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The ChIP assay was performed using the BeyoChIP™ ChIP Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, 1 × 107 CRC cells were prepared and then cross-linked with 1% formaldehyde for 15 min at room temperature. The cross-linking process was quenched by adding 0.125 M glycine. Chromatin was isolated with the lysis buffer provided in the kit. Afterwards, sonication was performed to shear the DNA to 200–1000 bp. Immunoprecipitation of cross-linked protein/DNA was performed by incubating anti-YBX1 antibody (Proteintech, IL, USA) or normal rabbit IgG (Proteintech, IL, USA) with sheared chromatin overnight at 4 °C followed by incubation with Protein A/G Magnetic Beads for 2 h. Subsequently, cross-links of protein/DNA were released with 5 M NaCl, and the DNA was purified using a PCR Clean Up Kit (Beyotime, Shanghai, China). Finally, the purified DNA was subjected to qRT–PCR using the P1-P9 primer sequences listed in Additional file 1: Table S4.
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6

ChIP-qPCR Assay for STAT3 Binding

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ChIP assays were performed using the BeyoChIP™ ChIP Assay Kit (P2080S, Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, MRC-5 cells (1 × 106) 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 10 sets of 30 s pulses (Bioruptor Pico, Liege, Belgium) to shear the DNA into fragments of approximately 200 to 1000 bp. Equal cell lysates were incubated with 1 μg of primary antibody against STAT3 overnight (Proteintech, China), and anti-IgG antibody (Beyotime, China) was used as a negative control. All the chromatin supernatants were then incubated with 80 μL of Protein A/G Magnetic beads/Salmon Sperm DNA for 1 h at 4 °C with rotation. The protein-DNA complexes were reversed and purified to obtain pure DNA. The qPCR following ChIP was performed with the specific primers listed below: 5′-CACTGGCCGCTGAAATTTAA-3′ and 5′-CACATACATCACGAACTCC-3′ for the human ATF6 promoter region (−1020/−909); 5′-GGAACGCTGCTGCATTATGTA-3′ and 5′-ACGTATGCTTCTAGGACCA-3′ for the human ATF6 promoter region (−1175/−1050).
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