ChIP was performed in H1299, H1299 KrasG13V and H460 KrasQ61H lung cancer cells manipulated for YY1 or ZNF322A. Lung cancer cells (1.5 × 106 cells) seeded in a 10 cm dish were cross-linked followed by preparation of nuclear lysates using Magna ChIPTM protein G Kit (Millipore). Nuclear lysates were sonicated to shear DNA to around 200~300 bp followed by immunoprecipitation for 16 h at 4 °C using IgG, anti-YY1 or anti-HA-ZNF322A antibody listed in Table S3 . Primers for PCR assay of ChIP samples and RT-qPCR reactions are listed in Table S2 .
Magna chip protein g kit
Manufactured by Merck Group
Sourced in United States
The Magna ChIPTM protein G Kit is a laboratory equipment used for chromatin immunoprecipitation (ChIP) assays. It provides a reliable and efficient method for the isolation and purification of protein-DNA complexes from cell or tissue samples. The kit includes magnetic protein G beads, buffers, and reagents necessary to perform the ChIP procedure.
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Lab products found in correlation
Solid 5500xl sequencer, by Thermo Fisher Scientific (2 mentions)
Anti nrf2 antibody, by GeneTex (1 mentions)
Anti p stat3 y705 antibody, by Cell Signaling Technology (1 mentions)
40 ti rotor, by Beckman Coulter (1 mentions)
Hif 2α, by GeneTex (1 mentions)
Anti ha antibody, by Abcam (1 mentions)
Oct4 antibody, by Abcam (1 mentions)
Anti sox17 antibody, by R&D Systems (1 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
11 protocols using magna chip protein g kit
1
ChIP Assay for Lung Cancer Cells
2
Chromatin Immunoprecipitation of CHD1L and HIF-2α
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The 786-O Cells (5 × 106) were cross-linked followed by the preparation of nuclear lysates using Magna ChIPTM protein G Kit (Millipore, Burlington, MA, USA). Nuclear lysates were sonicated to shear DNA to around 500 bp followed by immunoprecipitation for 16 h at 4 °C using IgG or anti-CHD1L or anti-HIF-2α antibody (Genetex, San Antonio, TX, USA). The levels of targeted genes in ChIP products were determined by RT-qPCR.
3
ChIP-qPCR Analysis of NRF2 Binding
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Cells (5 × 106) were cross-linked followed by preparation of nuclear lysates using Magna ChIPTM protein G Kit (Millipore, Burlington, MA, USA). Nuclear lysates were sonicated to shear DNA to around 500 bp followed by immunoprecipitation for 16 h at 4 °C using IgG or anti-NRF2 antibody (Genetex, San Antonio, TX, USA). The levels of targeted genes in ChIP products were determined by RT-qPCR. Primers used are listed in Table S1 .
4
Isolation and Analysis of Rab37-Specific Vesicles
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RAW264.7 cells (2.5×106) were sonicated and supernatants were obtained by centrifugation (3,000 g for 10 min at 4 °C) and vesicles were enriched from supernatants by high speed centrifugation (30,000 g for 60 min at 4 °C) using a 40-Ti rotor (Beckman, Duarte, CA, US). The vesicles-containing solution (500 μg) was incubated with anti-V5 antibody to isolate Rab37-specific vesicles and the IL-6 cargos in vesicles were analyzed by Western blot (Table S2 ). For ChIP assay, Jurkat T cells (5×106) were cross-linked to prepare nuclear lysates using Magna ChIPTM protein G Kit (Millipore, Cork, Ireland) followed by immunoprecipitation with 5 μg anti-p-STAT3(Y705) antibody (#9145, Cell Signaling, Danvers, MA, US). Q-PCR was carried out using ChIP products. The primer sequences are listed in Table S3 .
5
ChIP-qPCR Analysis of VEGFA
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The 786-O cells (5 × 106) were subjected to cross-linking, and then nuclear lysates were prepared using the Magna ChIPTM protein G Kit (Millipore, Burlington, MA, USA).
Following sonication of nuclear lysates to a length of approximately 500 base pairs, DNA was precipitated at 4 °Celsius for 16 h with either IgG or antibodies against KDM4B, H3K9me3, or HIF-2α (Genetex, San Antonio, TX, USA). RT-qPCR was utilized to quantify the levels of gene expression in the ChIP products. The following sequences were included in the list: VEGFA-promoter (5′->3′): GGCGGGTAGGTTTGAATC (sense), CGTATGCACTGTGGAGTC (antisense).
Following sonication of nuclear lysates to a length of approximately 500 base pairs, DNA was precipitated at 4 °Celsius for 16 h with either IgG or antibodies against KDM4B, H3K9me3, or HIF-2α (Genetex, San Antonio, TX, USA). RT-qPCR was utilized to quantify the levels of gene expression in the ChIP products. The following sequences were included in the list: VEGFA-promoter (5′->3′): GGCGGGTAGGTTTGAATC (sense), CGTATGCACTGTGGAGTC (antisense).
6
Genome-Wide Mapping of ZNF322A Binding Sites
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The chromatin-immunoprecipitation followed by deep sequencing (ChIP-seq) was performed in H460 cells expressing empty vector or ZNF322A expression vector. Cells were cross-linked and then lysed using Magna ChIPTM protein G Kit (Millipore, Bedford, MA, USA) for nuclear extraction. Nuclear lysates were sonicated and then immunoprecipitated with anti-HA antibody (Abcam, Cambridge, MA, USA). Purified ChIP DNA was prepared for fragment libraries, which were subjected to high-throughput sequencing using a SOLiDTM 5500xl sequencer (Applied Biosystems, Foster City, CA, USA). The raw reads were further analyzed using LifeScopeTM Genomic Analysis Software (version 2.5), and mapped to human genome (hg19) released from UCSC database [39 (link)]. The mapped profiles were analyzed using the ChIP-seq tool in CLC Genomics Workbench (version 4.9) with the human genome (hg19) as the default settings. Window size and false discovery rates were set to 200 bp and 5%, respectively. To determine the high confidence ZNF322A binding loci, the ChIP-region was identified by scanning the peaks with significantly higher read count in ZNF322A expressing cells compared to that in the control cells.
7
KLF9 ChIP-PCR protocol in HCT116 cells
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HCT116 cells were harvested, and the Chromatin immunoprecipitation (ChIP) experiments were performed using Magna ChIP protein G Kit (17-611, Millipore) according to the manufacturer's protocol. In brief, the cross-linked chromatin was sonicated into fragments, and cell lysates were immunoprecipitated using a KLF9 antibody. Finally, the precipitated DNA was analyzed by PCR with the primers listed in Table S3.
8
Oct4 ChIP-Seq Protocol for Identifying Binding Loci
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Stable Oct4 expressing cells or empty vector control (1 × 107 cells) were cross-linked with 1% formaldehyde, followed by preparation of nuclear lysates using Magna ChIP™ protein G Kit (Millipore Co., Billerica, MA, USA) according to the manufacturer's protocols. Nuclear lysates were sonicated to shear crosslinked DNA, followed by immunoprecipitation with anti-Oct4 antibody using the conditions described in Supplementary Table S3. Purified ChIP DNA was used for the preparation of fragment libraries, followed by high-throughput sequencing using a SOLiDTM 5500xl sequencer (Applied Biosystems, Foster City, CA, USA). We obtained about 24–29 million raw reads from vector and stable Oct4 cell line. The raw reads were further analyzed using LifeScopeTM Genomic Analysis Software (version 2.5) and mapped to human genome (hg19) released from UCSC database. To find the significant peak, the mapped profiles were analyzed using the ChIP-seq tool in CLC Genomics Workbench (version 4.9). Window size and false discovery rate (FDR) were set to 200 bp and 5%, respectively. To determine the high confidence Oct4 binding loci, the ChIP-region was identified by scanning the peaks with significantly higher read count in stable cells expressing Oct4 compared to those in the vector control cells. Oct4 ChIP-seq data can be viewed online under GEO accession number GSE58462.
9
Oct4 Chromatin Immunoprecipitation Protocol
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Empty vector control and Oct4 stably-overexpressed A549 cells (1 × 107 cells) were cross-linked with 1% formaldehyde for 10 min at 37 °C, followed by preparation of nuclear lysates using Magna ChIP™ protein G Kit (Millipore Co., Billerica, MA, USA). Nuclear lysates were sonicated to shear crosslinked DNA to around 300 ~ 500 bps using Covaris-S2 machine. Chromatin was immunoprecipitated with Oct4 antibody (1:100, # ab-19857, Abcam, Cambridge, UK). Purified chromatin-immunoprecipitated DNA was subjected to PCR analysis using primers for the lncRNA promoter and enhancer regions listed in Additional file 1 : Table S2.
10
Chromatin Immunoprecipitation Protocol for SOX17
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Cells were fixed with 1% formaldehyde and then quenched with glycine, followed by preparation of nuclear lysates using Magna ChIP™ protein G Kit (Millipore, Burlington, MA, USA). Nuclear lysates were sonicated to obtain DNA fragments around 500 bp and then subjected to immunoprecipitation for 16 h at 4 °C using 4 µg anti-SOX17 antibody (R&D Systems, Minneapolis, MN, USA) or normal IgG (negative control). The levels of targeted genes in ChIP products were determined by RT-qPCR. Primers are listed in Additional file 1 : Table S4.
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