ChIP assays were performed using EZ-Magna ChIP™ and One-Day Chromatin Immunoprecipitation kits (Millipore) according to previously described methods27 (link). Frozen tissues were cut into 1–3 mm pieces. A volume of 1 ml of PBS with protease inhibitors (Roche, Germany) was added per 100 mg of tissue. The DNA-protein complexes were cross-linked and then sheared to 200–1000 bp using a sonicator. The sonicated nuclear fractions were divided for input control and incubation with a negative control IgG or the STAT6 (D3H4) rabbit mAb (Cell Signaling Technology, USA). The antibody protein-DNA complex was then pulled down with magnetic beads coupled to anti-mouse IgG. The pellets were washed with a series of wash buffers, and the protein-DNA complexes were eluted with 100 μl of ChIP Elution Buffer and 1 μl of Proteinase K. The DNA was purified using spin columns. Finally, the Muc5ac promoter region was amplified using RT-PCR and quantitative real-time PCR with primers specific for the STAT6-binding elements of the MUC5AC promoter region (from −879 to +1 bp): forward primer: 5′-CCATCCCA GCAGACATGAAA-3′, reverse primer: 5′-CTATTAACCTCCTGAGC AACCC-3′. The specificity of each primer was confirmed by analyzing the melt curve and the amplification plot.
Ez magna chip
Manufactured by Merck Group
Sourced in United States
The EZ-Magna ChIP is a laboratory equipment product designed for chromatin immunoprecipitation (ChIP) assays. It facilitates the isolation and purification of DNA-protein complexes from cells or tissues. The core function of the EZ-Magna ChIP is to enable researchers to study gene regulation and epigenetic modifications.
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Lab products found in correlation
Formaldehyde, by Merck Group (2 mentions)
One day chromatin immunoprecipitation kit, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Anti notch3 antibody, by Cell Signaling Technology (1 mentions)
Anti h3k27ac antibody, by Abcam (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
H3k27ac, by Merck Group (1 mentions)
Chip grade protein a magnetic beads, by Merck Group (1 mentions)
Te buffer, by Merck Group (1 mentions)
Pab 069 050, by Diagenode (1 mentions)
Normal armenian hamster igg, by Santa Cruz Biotechnology (1 mentions)
9 protocols using ez magna chip
1
ChIP Assay for STAT6-Mediated MUC5AC Regulation
2
Chromatin Immunoprecipitation of Tet3 in mESCs
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Undifferentiated and day 3-differentiated mESCs were fixed in 1% formaldehyde (Sigma), harvested, lysed and sonicated as described for immuno-dot-blot analyses. ChIP assays were performed using the EZ-Magna ChIP™ (Millipore) kit according to manufacturer's instructions, using negative control IgG and antibodies to H3K27me3, H3K4me3 or Tet1. PCR amplification of the Tet3 promoter region (primer sequences shown in Table 1 ) was performed in 25 μl reaction volumes using Q5® high fidelity DNA polymerase, according to the manufacturer's instructions, with added Enhancer buffer. H3K27me3 and H3K4me3 were amplified for 28 cycles and Tet1 for 30 cycles, at a 66°C annealing temperature and 30 s extension time.
3
Chromatin Immunoprecipitation Sequencing
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ChIP was performed using EZ-Magna ChIP (Millipore) according to the manufacturers protocol. In brief, cells were fixed, sheared by sonication and immunoprecipitated with rabbit IgG control (supplied in the kit), anti-NOTCH3 antibody (Cell Signaling, #2889), or anti-H3K27Ac antibody (Abcam, ab4729). Libraries were constructed using an NEB NEXT kit, and sequencing was done on an Illumina Hiseq2000 instrument. ChIP-seq data are available through the Gene Expression Omnibus (accession number GSE104262)
4
ChIP-qPCR Protocol for Histone Modifications
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ChIP was performed with EZ-Magna ChIP (catalog #17-408; Millipore, Burlington, MA) with specific immunoprecipitating antibodies including H3K27Me3 or H3K27Ac, with the negative control normal rabbit IgG as previously described.7 Cells were cross-linked with 1% formaldehyde, after which they were lysed and subjected to sonication, then sheared cross-linked to fragment DNA. After centrifugation, the supernatant was diluted in ChIP buffer. Samples subsequently were incubated with 5 μg antibody and ChIP-grade Protein A magnetic beads (Millipore) overnight at 4°C. After extensive washing (Low Salt, High Salt, LiCI Immune Complex Wash Buffer, and TE Buffer; Sigma-Aldrich Inc, St. Louis, MO), chromatin was eluted, and DNA was purified and analyzed by qPCR. Fold enrichment was calculated by first normalizing ChIP-qPCR to input DNA of the target gene as a percentage of input. This subsequently was normalized to the percentage input of a negative control gene region (intergenic region of 36Me3-hCh19) to correct for experimental variation. H3K27Ac (4279), H3K27me3 (6002), and isotype-specific IgG control (2027) were purchased from Abcam (Cambridge, MA).
5
TDP-43 Chromatin Immunoprecipitation in SH-SY5Y Cells
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SH-SY5Y cells (4 × 106) were plated 24 h before transduction, and infected by using viruses encoding for TDP WT at a multiplicity of 30 pfu/cell. After 24 h, cells were harvested and chromatin immunoprecipitation was performed using EZ-Magna ChIP™ (Millipore), according to the manufacturer’s protocol.
Each immunoprecipitated (IP) reaction was performed using about 1 × 106 cells equivalents of chromatin. The antibodies used for immunoprecipitation were the following: TARDBP Polyclonal Antibody (Proteintech_10782-2-AP) and Normal Rabbit Ig (reagent supplied) as negative control. Purified chromatin was eluted and DNA fragments were used for qPCR (S2). The results were normalized using the Fold Enrichment Method (ChIP signals were divided by the no-antibody signals, representing the ChIP signal as the fold increase in signal relative to the background signal). **p < 0,01 Student’s t test.
Each immunoprecipitated (IP) reaction was performed using about 1 × 106 cells equivalents of chromatin. The antibodies used for immunoprecipitation were the following: TARDBP Polyclonal Antibody (Proteintech_10782-2-AP) and Normal Rabbit Ig (reagent supplied) as negative control. Purified chromatin was eluted and DNA fragments were used for qPCR (S2). The results were normalized using the Fold Enrichment Method (ChIP signals were divided by the no-antibody signals, representing the ChIP signal as the fold increase in signal relative to the background signal). **p < 0,01 Student’s t test.
6
Chromatin Immunoprecipitation Protocol
7
MUC1-CT Regulation of PTGS2 Expression
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Cells grown to near 80% confluence were cross-linked with formaldehyde (Sigma) at room temperature for 10 minutes. Cross-linked chromatin prepared with a commercial chromatin immunoprecipitation (ChIP) assay kit (EZ-Magna ChIP; Millipore) was immunoprecipitated with normal Armenian hamster IgG (1:20) (Santa Cruz Biotechnology), anti–MUC1-CT antibody (CT2) (1:15), and anti–NF-κB p65 (1:20) antibody. IgG was used as a negative control for the immunoprecipitation step. Input DNA (2%) and DNA isolated from the precipitated chromatin were amplified by polymerase chain reaction (PCR) using mouse- or human-specific primers flanking the promoter region containing the NF-κB–binding site (ChIP region I) or distant sites from the promoter region (ChIP region II). Chromatin immunoprecipitation region II was used as a negative control to evaluate specificity of association between MUC1-CT and NF-κB p65 to the promoter region of Ptgs2/PTGS2 gene. Sequence of the primers is available upon request.
8
ChIP Assay for T98G and U251 Cells
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T98G and U251 cells were used for CHIP assay. EZ-Magna ChIP (Millipore) kit was recommended in ChIP assay. The experiment was briefly described as follows. First, 1% formaldehyde solution was applied to the cells to induce cross-linking. Then the cross-linking was quenched with 140 mM glycine. The acquired nucleic acid protein complex was then lysed to 200-500 bp DNA fragments and immunoprecipitation of the experimental antibody or IgG. After 4°C overnight, the cross-linking of DNA was removed, and target fragment level was assayed via qRT-PCR. The primers used in the assay were in Table S2 . The antibody information used in the assay was in Table S3 .
9
WT1 Binding Sites in CCNA1 Promoter
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A total of 3 kb of the CCNA1 promoter gene sequence was downloaded from the public database, Ensembl (http://useast.ensembl.org/) and potential WT1 binding sites (GnGGGnG; Matrix Family Library Version 9.3) were queried using MatInspector Software (https://www. genomatix.de/). The binding function of these predicted sites was tested in WT1-transfected K562 cells using ChIP assays (Millipore EZ-Magna ChIP, Temecula, CA, USA), following manufacturer's instructions. WT1-bound fragments were immunoprecipitated using a mix of the WT1 antibodies (2.5 µg each of N18 and C19; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or 1 µg mouse IgG fraction provided with the kit as a negative control. WT1 bound chromatin was amplified by end-point PCR using primers specific for each of the three predicted sites (Table IIC) and analyzed by electrophoresis in ethidium bromide-containing agarose gels. The specificity of WT1 binding was tested by the amplification of immunoprecipitated chromatin using negative control primers located approximately 1.5 kb 5' of the third WT1 site and lacking any potential WT1 binding sites.
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