The ChIP kit was purchased from Cell Signaling (SimpleChIP Plus Chromatin IP kit, #9005) and the experiments were performed according to the protocol provided by Cell Signaling Technology. Briefly, cells grew in charcoal stripped FBS containing media up to 90% confluence prior to crosslink using 37% formaldehyde for 10 minutes at room temperature. Nuclei/chromatin was digested and sonicated after 3 sets of 20-second pulse and 3 sets of 30-second on wet ice between pulses (Branson Digital Sonifier 450). Chromatin lysates were incubated with anti-CREB antibody (#4820, Cell Signaling) or anti-IgG (#2729, Cell Signaling) at 4°C with rotation overnight. The eluted DNA products were subsequently utilized for real-time qPCR assay. Primers for detecting FN1-exon1 are available in Supplementary Table 6 . The details of the PCR reaction program are: initial denaturation: 95°C, 3 minutes; Denature: 95°C, 15 seconds; Anneal & Extension: 60°C, 60 seconds; Repeat step Denature and Anneal & Extension for a total of 40 cycles.
Simplechip plus chromatin ip kit
Manufactured by Cell Signaling Technology
Sourced in United States
The SimpleChIP Plus Chromatin IP kit is a laboratory instrument designed for the extraction and purification of chromatin from cells. It facilitates the immunoprecipitation of protein-DNA complexes, enabling the study of gene regulation and epigenetic mechanisms.
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2 protocols using simplechip plus chromatin ip kit
1
ChIP Assay for CREB Binding
2
Chromatin Immunoprecipitation Assay for aPKC-ι Promoter
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Chromatin immunoprecipitation (Ch-IP) assays were performed using SimpleChIP Plus Chromatin IP Kit (Cell Signaling, Massachusetts, USA) according to the manufacturer’s instructions. Hepatoma cells were transfected with recombinant plasmids to overexpress aPKC-ι promoter (pGL3-basic-pkci; WT group) or aPKC-ι promoter plasmids with a mutated Elk1 binding site (pGL3-basic-pkci-kt; MUT group). Cells were fixed with formaldehyde to crosslink DNA-proteins, chromatin was sheared using Microson Ultrasonic Cell Disruptor (Misonix, New York, USA) and incubated with antibodies, and IPs were bound to Protein G magnetic beads. The protein-DNA cross-link was reversed, DNA was purified, and enrichment of DNA sequences was detected using PCR and primers (listed in Supplementary File 1 ). Cell lysates without immunoprecipitation were analyzed with PCR as the positive control (Input), and DNA precipitated by IgG were used as the negative control.
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