Cells were lysed in cold NP-40 lysis buffer (1% vol/vol NP-40, 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl) supplemented with Halt™ Protease and Phosphatase inhibitor Cocktail. HA-tagged proteins were immunoprecipitated with anti-HA and ChIP-Grade Protein G Magnetic Beads (Cell Signaling Technology). Flag-tagged proteins were immunoprecipitated with Anti-FLAG M2 Magnetic Beads (Sigma-Aldrich). For the endogenous interaction assay, macrophages were lysed with NP-40 lysis buffer with protease inhibitors. The cell lysates were incubated overnight at 4 °C with NEMO antibody and ChIP-Grade Protein G Magnetic Beads (Cell Signaling Technology).
Chip grade protein g magnetic beads
Manufactured by Cell Signaling Technology
Sourced in United States
ChIP-Grade Protein G Magnetic Beads are designed for the efficient immunoprecipitation of protein-DNA complexes in chromatin immunoprecipitation (ChIP) assays. The beads are coated with recombinant Protein G, which has a high affinity for the Fc region of many antibody isotypes, enabling the capture of antibody-bound protein-DNA complexes. The magnetic properties of the beads allow for easy separation and washing of the captured complexes.
Automatically generated - may contain errors
Lab products found in correlation
Normal rabbit igg, by Cell Signaling Technology (8 mentions)
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (7 mentions)
Simplechip plus enzymatic chromatin ip kit, by Cell Signaling Technology (4 mentions)
Anti pparγ 16643 1 ap, by Proteintech (2 mentions)
Anti flag m2, by Merck Group (2 mentions)
Magnetic separation rack, by Thermo Fisher Scientific (2 mentions)
Micrococcal nuclease, by Cell Signaling Technology (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Histone h3 antibody, by Cell Signaling Technology (2 mentions)
Proteinase k, by Cell Signaling Technology (2 mentions)
Anti flag m2 magnetic bead, by Merck Group (2 mentions)
Simplechip plus sonication chromatin ip kit, by Cell Signaling Technology (2 mentions)
Ab32358, by Abcam (1 mentions)
Miseq next generation sequencer, by Illumina (1 mentions)
Thruplex dna seq kit, by Takara Bio (1 mentions)
Anti histone h3k27me3, by Abcam (1 mentions)
Anti runx2, by Cell Signaling Technology (1 mentions)
Anti histone h3k27ac, by Active Motif (1 mentions)
Anti histone h3k4me3, by Abcam (1 mentions)
Anti runx3, by Cell Signaling Technology (1 mentions)
40 protocols using chip grade protein g magnetic beads
1
Immunoprecipitation of Tagged and Endogenous Proteins
2
Chromatin Immunoprecipitation and qPCR Analysis
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Chromatin immunoprecipitation quantitative PCR (ChIP‐qPCR) was performed according to a previous report with some adjustments.39 Briefly, harvested cells were fixed followed by glycine treatment. Collected cells were washed and treated with a lysis buffer supplemented with a protein inhibitor cocktail (#P2714, Sigma). Then, the mixture underwent sonication to produce 200–400 bp chromatin segments. After centrifugation, the obtained supernatant was pre‐blocked with ChIP‐grade Protein G Magnetic Beads (#9006, Cell Signaling Technology) and incubated with anti‐p65 antibody (#8242, Cell Signaling Technology) and regular rabbit IgG overnight at 4°C. The mixture was incubated with ChIP‐grade Protein G Magnetic Beads (#9006, Cell Signaling Technology) for 2 h at 4°C. The magnetic beads were collected and washed. The collected beads were dissolved in elution buffer and treated with NaCl, RNase A and proteinase K. The same volume of mixture of Phenol:Chloroform:Isoamyl alcohol (25:24:1) (#A0417977, ACROS, Fair Lawn, NJ, USA) was added, vortexed and incubated for 20 min at RT. The supernatant was collected. A double volume of ethanol was added to the supernatant and placed at −20°C overnight. Finally, the DNA was extracted. Obtained DNA was used for qRT‐PCR. The primers are listed in Table1 .
3
ChIP Assay for CEBPB and CEBPD
4
ChIP-qPCR Analysis of PC4 and STAT3
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MDA-MB-231 cells were conducted ChIP assay with the SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads) (#9003; Cell Signaling Technology) according to the manufacturer’s instructions. Chromatin fragments were immunoprecipitated with anti-PC4 (NB10059774; Novus Biologicals), or rabbit IgG (#2729; Cell Signaling Technology) coupled with ChIP Grade Protein G Magnetic Beads (#9006; Cell Signaling Technology). After DNA purification, quantitative PCR was performed using primers (Additional file 1 : Table S2). The fold enrichment was calculated by normalizing samples of anti-TCF-1 or p-STAT3 to normal rabbit IgG controls.
5
ChIP-Seq of Mesenchymal Chondrosarcoma Cells
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ChIP-Seq was carried out using the method previously described using a biological duplicate (58 (link)). Briefly, 5 × 106 mesenchymal chondrosarcoma cells per immunoprecipitation were cross-linked with 1% formaldehyde for 10 minutes at room temperature. Chromatin was sheared in SDS lysis buffer containing 1% SDS, 10 mM EDTA, and 50 mM Tris, pH 8.0, to an average size of 400–500 bp using a Covaris S220 sonicator for 15 minutes. ChIP was carried out with 5 μg anti-FLAG (Sigma-Aldrich), anti-histone H3K27ac (Active Motif), anti-histone H3K27Me3 (Abcam), anti-histone H3K4Me3 (Abcam), anti-Runx2 (Cell Signaling), or anti-Runx3 (Cell Signaling) antibodies. The antibody-bound protein/DNA complexes were immunoprecipitated using ChIP grade protein G magnetic beads (Cell Signaling).
Immunoprecipitated DNA was then purified and subjected to secondary sonication to an average size of 150–350 bp. Libraries were prepared according to instructions accompanying the ThruPLEX DNA-Seq kit (Rubicon Genomics). The ChIP DNA was end modified and adapters were ligated. DNA was PCR amplified with Illumina primers, and Illumina-compatible indexes were added. The library fragments of approximately 300-500 bp were band-isolated from an agarose gel. The purified DNA was sequenced on an Illumina MiSeq next-generation sequencer following the manufacturer protocols.
Immunoprecipitated DNA was then purified and subjected to secondary sonication to an average size of 150–350 bp. Libraries were prepared according to instructions accompanying the ThruPLEX DNA-Seq kit (Rubicon Genomics). The ChIP DNA was end modified and adapters were ligated. DNA was PCR amplified with Illumina primers, and Illumina-compatible indexes were added. The library fragments of approximately 300-500 bp were band-isolated from an agarose gel. The purified DNA was sequenced on an Illumina MiSeq next-generation sequencer following the manufacturer protocols.
6
ChIP Assay for PPARs in MM Cells
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ChIP assays were performed as previously described. Briefly, MM cells were collected, cross-linked with 1% formaldehyde, and centrifuged. The resulting pellets were sonicated, and the chromatin solution was precleared with 30 μL of ChIP-grade protein G magnetic beads (#9006; Cell Signaling Technology, Danvers, MA, USA). The soluble fraction was collected, and the chromatin beads were incubated with positive control histone H3 rabbit (#4620; Cell Signaling), normal rabbit IgG (#2729; Cell Signaling), anti-PPARα (ab227074; Abcam), anti-PPARγ (16643-1-AP; Proteintech Rosemont, IL, USA), and anti-FLAG M2 (F1804; Sigma; for PPARβ/δ with a FLAG tag) antibodies at 4 °C overnight. The ChIP-enriched DNA was analyzed by quantitative PCR using the CRBN promoter primers as follows: forward: 5′-TCCCCTGTCACCTTCAAAAC-3′ and reverse: 5′-TGCCTTGTGAGTCTGACACC-3′. The enrichment of the CRBN promoter regions was assessed relative to the input DNA, followed by normalization to the respective control IgG values. Percent input = 4% × 2(C[T] 4% input sample- − C[T] IP sample).
7
ChIP Assay of MDA-MB-231 Cell Epigenetics
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ChIP assay was performed using the ChIP-Grade Protein G Magnetic Beads (Cell Signaling Technology, Inc., Danvers, CO, USA). Briefly, MDA-MB-231 cells were sonicated after treatment with 1% PFA, neutralized, and resuspended in SDS lysis solution (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.0, and PMSF) for chromatin fragmentation. Sheared chromatin was diluted and normal IgG was used for immunoprecipitation. Reversing the crosslinking and digestion with proteinase K were used to extract DNA from immunoprecipitates, which was subsequently PCR amplified. The ChIP qPCR data were analyzed as previously described [20 (link)].
8
ChIP Assay Protocol for Transcription Factors
9
ChIP Assay of STAT4 and STAT5 Binding
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Chromatin immunoprecipitation (ChIP) assay was done according to the manufacturer’s instructions. Briefly, naïve CD8+ T cells were purified from Stat1+/+ or Stat1−/− mice and cultured with IL-7 (1 ng/ml) and IFN-β (10 ng/ml) for 2 days. Cells were fixed with 37% of formaldehyde, resuspended in the supplied ChIP buffer, and lysed by sonication. Abs for STAT4 (C46B10; Cell Signaling Technology) and STAT5 (D2O6Y; Cell Signaling Technology) were added to the ChIP sample, followed by incubation of the samples overnight at 4°C with rotation. ChIP-grade protein G magnetic beads (Cell Signaling Technology) were added to the samples, and the tubes were placed in the Magnetic Separation Rack (Invitrogen) to isolate the Ab-bound chromatins. Chromatins were eluted from the Ab/magnetic beads by elution buffer at 65°C with vortexing and purified in the spin column. Real-time polymerase chain reaction (PCR) was done with purified DNA and primers for the predicted RagD promoter or enhancer sites, which can be bound by STAT TFs by JASPAR2020 (51 (link)): rragd -864 primer: 5′-GAAAGCAGCCACCCAGTTAG-3′ (forward) and 5′-TAGGGGTCCTCGGAAAAATC-3′ (reverse); rragd -337 primer: 5′-CCGAGGGCGTTATACACTTT-3′ (forward) and 5′-GACATGTTTCCGGAGAAGTCA-3′ (reverse); and rragd -222 primer: 5′-TCACGAGACATGTGACTTCTCC-3′ (forward) and 5′-AGCAGCTGCCTCCTAAAGTG-3′ (reverse).
10
Chromatin Immunoprecipitation Protocol
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A SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology) was used to carry out the experiment according to the manufacturer’s instructions. We first added formaldehyde to the cell culture dishes to crosslink the protein with DNA. After that, glycine was added to terminate the crosslinking and the cells were scraped off the plate. Then, the DNA was digested to a length of about 150–900 bp, and the nucleus was completely cleaved using an ultrasonic crusher. Abs were added to the lysate at 4°C overnight. The Abs used in this paper include Normal Rabbit IgG (Cell Signaling Technology, 2729), Histone H3 Rabbit mAb (Cell Signaling Technology, 4620), and anti–β-catenin (Abcam, ab32572). ChIP-Grade Protein G Magnetic Beads (Cell Signaling Technology, 9006) were added to the sample and incubated at 4°C for 2 hours. The chromatin was eluted by gently swirling, mixing, and incubating at 65°C for 30 minutes, followed by adding NaCl and proteinase K at 65°C for 2 hours. Finally, DNA was purified and detected by qPCR.
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