The assay was performed using ChIP-IT Express Enzymatic Kit (Active Motif) as described earlier (22 (link)). In brief, cells (5 × 106) were cultured in 150 mm dishes to 60 to 70% confluence and treated with different concentrations of DHT for 24 h. Thereafter, cells were incubated with 1.0% formaldehyde (Sigma-Aldrich) for 10 min to cross-link DNA and proteins and scraped in an ice-cold cell-scraping solution supplemented with protease inhibitors. The cross-linked chromatin (DNA–protein complexes) was enzymatically sheared using ChIP-IT Express Enzymatic Kit (Active Motif) and subjected to immunoprecipitation using protein G magnetic beads using anti-AR or normal rabbit IgG (control) antibodies. Magnetic beads were washed and cross-linking reversed using reverse cross-linking buffer followed by proteinase K digestion and DNA purification. The immunoprecipitated DNA was purified and subjected to qRT-PCR using specific primer sets as mentioned in Table S2 .
Chip it express enzymatic kit
Manufactured by Active Motif
Sourced in United States
The ChIP-IT Express Enzymatic kit is a tool used for chromatin immunoprecipitation (ChIP) experiments. It provides a streamlined protocol for preparing chromatin from mammalian cells and performing the immunoprecipitation step.
Automatically generated - may contain errors
Lab products found in correlation
Qiaquick pcr purification kit, by Qiagen (5 mentions)
Chromatin ip dna purification kit, by Active Motif (4 mentions)
Normal rabbit igg, by Cell Signaling Technology (4 mentions)
Bioruptor pico, by Diagenode (3 mentions)
Fugene hd, by Promega (3 mentions)
Re chip it kit, by Active Motif (2 mentions)
Chip it high sensitivity kit, by Active Motif (2 mentions)
Ideal chip qpcr kit, by Diagenode (2 mentions)
Rabbit anti sox9 antibody, by Abcam (2 mentions)
Anti flag or igg control antibodies, by Thermo Fisher Scientific (2 mentions)
Mouse anti flag, by Merck Group (2 mentions)
Sybr green, by Bio-Rad (2 mentions)
Lightcycler 480 2, by Roche (2 mentions)
Ezview red anti flag m2 affinity gel, by Merck Group (2 mentions)
Sybr select master mix, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Beyotime (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
123 protocols using chip it express enzymatic kit
1
ChIP-IT Express Enzymatic Chromatin Immunoprecipitation
2
Quantitative ChIP-qPCR Analysis of Histone Modifications
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ChIP assay was performed using the ChIP‐IT Express Enzymatic Kit (Active Motif) according to the manufacturer’s protocol. Briefly, the cells were fixed by 1% formaldehyde. After sonication, the chromatin was immunoprecipitated with antibodies against different histone‐PTM or mouse/rabbit IgG for 16 hours. The DNA purification was performed using the ChIP‐IT Express Enzymatic Kit (Active Motif). A total of 5 μL of ChIP‐enriched DNA was used as a template for quantitative PCR (qPCR) amplification with designated primers (Table S2 ). The cycle threshold (Ct) values from each run were averaged per tissue or cell, and the ΔΔCT method was then used for the data analysis, in which the value of target DNA fragments that were enriched in each tissue or cell was normalized to the value of the 5% input DNA of each sample. The qPCR data were expressed as the mean ± SD. The ChIP‐qPCR assay was repeated twice to confirm the reproducibility of the results. The results were represented as fold changes compared with the control group.
3
Chromatin Immunoprecipitation to Analyze HNF4α Binding
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Chromatin was extracted from 20 million Hepa 1.6 or NIH3T3 cells with ChIP-IT Express Enzymatic kit (Active Motif), according to the manufacturer’s recommendations. Every ChIP reaction was performed with 5 μg of mouse anti-HNF4α antibodies (H1415, Life Technologies). Immunoprecipitated DNA and Input DNA were purified with Chromatin IP DNA Purification Kit (Active Motif). It was subsequently analyzed by qPCR to measure the relative enrichment of the fragments of interest in the total input of ChIP DNA fragments. The negative control ChIP primers were designed to amplify fragments downstream of the Agxt2 predicted transcription start sites. The positive control primers were designed to amplify the region of hepatocyte nuclear factor 1 alpha (Hnf1a) promoter, which has been shown previously to comprise an active HNF4A binding site28 (link). Murine fibroblast cell line NIH 3T3 was used as an additional negative control. ChIP-IT Control qPCR Kit for Mouse (Active Motif) was used as an additional control.
4
Chromatin Immunoprecipitation of CCL19 Promoter
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Cells and tissues protein were lysed with RIPA buffer (Beyotime, Jiangsu, China). All protein was run on a 10% SDS-polyacrylamide gel. Primary antibody against CCL19, Collagen 1, IL-6, IRF-1 and β-actin were purchased from Affinity Bioscience (Jiangsu, China), with β-actin as an internal control. Odyssey Infrared Imaging System (Odyssey CLx, Biosciences, USA) was used to detect immunoreactivity.
Chromatin Immunoprecipitation (ChIP) ChIP experiment uses ChIP-IT Express Enzymatic kit, purchased from Active Motif company in the United States, the experimental steps are carried out in strict accordance with the product instructions: DNA purification and qPCR analysis. The CCL19 promoter region, which contains two IRF-1 binding sites was detected. The experimental samples are analyzed by Input samples, and the data are quantized as the percentage of Input.
5
ChIP Assay for TSPYL5 Gene
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ChIP assays were performed using a ChIP-IT Express Enzymatic Kit (Active Motif, Carlsbad, CA, USA). Briefly, the cells were fixed with formaldehyde, and then the cross-linked chromatin was digested using an enzymatic shearing cocktail. A portion of the optimally sheared chromatin was retained as a control “input DNA” in the subsequent PCRs. The remaining chromatin was precipitated by incubation with antibodies against HA and TSPY1. PCR was performed to amplify the target region of the TSPYL5 gene. The primers for PCRs are listed in Supplementary Table S2 .
6
Chromatin Immunoprecipitation Assay for Histone Acetylation
7
ChIP-qPCR Analysis of LIMD1 Promoter
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ChIP was performed in 293 cells as described in our previous publication [55 (link)], with the use of ChIP-IT Express Enzymatic kit (Active Motif). qPCR was performed with the human LIMD1 promoter EICE primers: 5′- AA GGCTGCGGCAAGGGGCCG-3′ (forward) and 5′-CACC AGGCCTGACTCCTTGG-3′ (reverse), and the NFκB- binding site primers: 5′-TGCGCGCAGGCACAACG AG-3′ (forward) and 5′- CGTGTCACCCATGGCTGG-3′ (reverse).
8
Chromatin Immunoprecipitation (ChIP) Protocol
9
PARP1 Binding Site Identification in CXCL12 Promoter
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T265-2c cells were plated in 100mm dishes at densities described above in the Cell Culture section and drug treatments were performed after 24h. To account for loss of total cell number resulting from cytotoxic effects of BH3 mimetic treatment, twice as many 100mm dishes were used for each treatment condition versus untreated and each dish of respective cells was combined after fixation. ChIP using antibodies against PARP1 (Santa Cruz H-250 [sc-7150]) (12μg/ml) or rabbit IgG (Santa Cruz sc-2027) (12μg/ml) was performed using the ChIP-IT Express Enzymatic kit from Active Motif (Carlsbad, CA #53009) per the manufacturer's instructions. DNA was amplified using primers flanking the putative PARP1 [“A/GNNA/TCAAA” [53 (link)] binding site within the CXCL12 promoter. Negative control primers were obtained from Active Motif (#103708).
Primer sequence:
Forward: 5′-GAATCTCCCGTCCCACTCC-3′
Reverse: 5′-GCCGAGCCTCAGTTTCCT-3′
Primer sequence:
Forward: 5′-GAATCTCCCGTCCCACTCC-3′
Reverse: 5′-GCCGAGCCTCAGTTTCCT-3′
10
ChIP Analysis of STAT3 Binding on TXNIP Promoter
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