U937-derived macrophages treated without or with IL-11 (25 ng/mL) were harvested for ChIP using Pierce Magnetic ChIP kit (ThermoFisher Scientific, Cat# 26157) following manufacturer’s protocol. Briefly, cells (2 × 106) were cross-linked using 1% formaldehyde for 10 min and treated with glycine for 5 min at room temperature. Cells were lysed and DNA was pre-digested by MNase at 37°C for 15 min. DNA were sheared to fragments of 300–500 bp in size by Q800R3 Sonicator at 20% amplitude for 4 min with 10 s on/20 s off cycles (Qsonica). DNA fragments were incubated with anti-pSTAT3 antibody (Cell Signaling Technology, Cat# 9131) or normal rabbit IgG (Pierce Magnetic ChIP kit, ThermoFisher Scientific, Cat# 26157) at 4°C for 16 h. After proteinase K digestion, immunoprecipitated DNA fragments were eluted and amplified using quantitative real-time PCR (Roche). Antibody signals were normalized to the input signals and Student’s t test was performed to compare the results. Primer sequences were listed in Table S5 .
Pierce magnetic chip kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce Magnetic ChIP Kit is a tool designed for chromatin immunoprecipitation (ChIP) experiments. It utilizes magnetic beads coated with protein A or protein G to capture antibody-bound chromatin fragments, enabling efficient isolation and purification of DNA sequences associated with specific proteins of interest.
Automatically generated - may contain errors
Lab products found in correlation
Normal rabbit igg, by Thermo Fisher Scientific (4 mentions)
Q800r3 sonicator, by Qsonica (3 mentions)
Dual luciferase reporter assay system, by Promega (3 mentions)
Formaldehyde, by Merck Group (3 mentions)
Anti smad4 antibody, by R&D Systems (2 mentions)
Anti lrp1 icd antibody, by Abcam (2 mentions)
Normal goat igg antibody, by Abcam (2 mentions)
Quantstudio 5, by Thermo Fisher Scientific (2 mentions)
Bioruptor pico, by Diagenode (2 mentions)
Dynabead, by Thermo Fisher Scientific (2 mentions)
Column method, by Thermo Fisher Scientific (2 mentions)
Ab9110, by Abcam (2 mentions)
H3k27ac, by Cell Signaling Technology (2 mentions)
H3k9me3, by Abcam (2 mentions)
I 1000, by Vector Laboratories (2 mentions)
Anti p65 antibody and normal igg, by MultiSciences Biotech (2 mentions)
Sybr green pcr master mix, by Qiagen (2 mentions)
Biorupter, by Diagenode (2 mentions)
Anti ctcf antibody, by Abcam (2 mentions)
Ab58310, by Abcam (2 mentions)
175 protocols using pierce magnetic chip kit
1
ChIP-qPCR Analysis of pSTAT3 in IL-11-Treated Macrophages
2
ChIP-qPCR Protocol for Protein-DNA Interactions
3
Dual Luciferase Assay and ChIP Analysis
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A dual luciferase reporter assay system was used to measure the promoter activity according to manufacturer's instruction (Promega Corporation). The Renilla luciferase driven by TK minimal promoter was used as an internal control to normalize the transfection efficiency. Luciferase activity was measured 48 h post-transfection by using an EnSpire® multimode plate reader (Perkin Elmer, Inc.). ChIP assay was carried out according to the manufacturer's instructions (Pierce™ magnetic ChIP kit; Thermo Fisher Scientific, Inc. cat. no. 25157). Briefly, 2×106 cells were transfected with Myc-tag EHF expression construct and cross-linked in 1% formaldehyde at 37°C for 10 min and lysed in SDS lysis buffer (Pierce magnetic ChIP kit; Thermo Fisher Scientific, Inc.) supplemented with protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA) for 10 min on ice. The lysate was sonicated and precleared with protein A agarose/salmon sperm DNA prior to immunoprecipitation with 1 µg of anti-Myc Tag antibody (cat. no. 05-724; Clone 4A6; EMD Millipore) at 4°C overnight. Following wash and elution steps, cross-links were reversed at 65°C for 4 h. The DNA in the immunoprecipitated samples was purified by proteinase K digestion followed by DNA purification. An aliquot of purified DNA was analyzed by quantitative PCR with specific primers.
4
ChIP-qPCR Protocol for Protein-DNA Interactions
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ChIP assays were performed using Pierce Magnetic ChIP Kit according to the manufacturer’s protocol (ThermoFisher Scientific, Cat# 26157). All reagents used were included in the kit. Briefly, 4 × 106 cells were cross-linked using 1% paraformaldehyde for 5 min and reactions were quenched by glycine for 5 min at room temperature. After lysis and MNase digestion, DNA fragments in the cell lysates were sheared to size 300–500 bp by the Q800R3 Sonicator (20% amplitude, 10 s on, 20 s off for 8 min) (Qsonica). Then, cell lysates were incubated with an anti-SMAD4 antibody (R&D Systems, Cat# AF2097), anti-LRP1-ICD antibody (Abcam, Cat# ab92544), normal goat IgG antibody (Abcam, Cat# ab37373) or normal rabbit IgG antibody (Pierce Magnetic ChIP Kit, ThermoFisher Scientific, Cat# 26157) overnight at 4°C. The immunoprecipitated genomic DNA fragments were eluted and amplified using quantitative real-time PCR. Specific antibody signals were normalized to the input signals. Quantitative real-time PCR results were compared statistically using Student’s t test. Primers used for qPCR analysis are listed in Table S4 .
5
Chromatin Immunoprecipitation of HK-2 Cells
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Chromatin immunoprecipitation was performed using Pierce Magnetic ChIP Kit (Thermo Fisher, 26157). The HK-2 cells were crosslinked using formaldehyde with a final concentration of 1%, and the number of cells required 2 × 106.
6
ChIP Assay in Pancreatic Cancer Cells
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ChIP assays were performed in pancreatic cancer cells transfected with shHDAC5 and Flag-HDAC5 using the Pierce Magnetic ChIP Kit (Code No. 26157, Thermo Fisher Scientific) according to the manufacturer's instructions. Protein G magnetic beads and specific antibody were used for IP of chromatin, about 5μg antibodies were used for 25 μg chromatin. In addition, normal rabbit IgG was served as negative control. Finally, PCR Kit (Promega) was performed to purified input and immunoprecipitated DNA. After purification, qPCR was used to check the promoters and enhancers of the DNA samples. The sequences of the primers were provided in Supplementary Table S1.The antibodies used were provided in Supplementary Table S2.
7
Quantitative ChIP-qPCR Analysis
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Cells (5 × 107) were crosslinked with 1% formaldehyde (Sigma-Aldrich; #252549) to covalently stabilize protein-DNA complexes. The cells were lysed in the lysis buffer provided in the Pierce Magnetic ChIP Kit (ThermoFisher Scientific; #26157), then sonicated to shear the DNA to lengths of approximately 500 bp. The resulting products were immunoprecipitated using anti-CtIP, anti-CtBP1, anti-CtBP2, anti-JUN, anti-FOS, and IgG. The input and output DNA were used for qRT-PCR analyses to measure protein occupancies on the promoters of genes, using the primers listed in Table S4.
8
ChIP-qPCR for Sp1 in NSCs
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Chromatin immunoprecipitation (ChIP) for the primary NSCs was performed using a Pierce Magnetic ChIP Kit (Thermo Fisher Scientific, United States) according to the manufacturer’s instructions. An anti-Sp1 antibody suitable for ChIP (1:100 dilution; Cell Signaling Technology; United States; Cat# 9389; AB_11220235 ) or rabbit IgG (1:250 dilution; Thermo Fisher Scientific; United States; Cat# 31887; AB_2532177 ) was use. qRT-PCR was performed to obtain quantitative data using 2 × Taq Plus Master Mix (Vazyme, China), and TB Green Premix Ex Taq II (TaKaRa, China). The enrichment at the cdkn1b promoter region was normalized to the amount of the total input. The Primers for the cdkn1b promoters can be found in the (Supplementary Table 1 ).
9
Chromatin Immunoprecipitation and Real-Time PCR
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ChIP was performed with the Pierce Magnetic ChIP Kit (Thermo Fisher Scientific) following the manufacturer instructions. A total of 3 µg of p53 antibody (Cell Signaling Technology; #2524) were used per IP.
Total cellular RNA was extracted using the RNeasy Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. One-step reverse transcription and real-time PCR were performed with a Quantstudio 5 (Thermo Fisher Scientific) using QuantiTect SYBR Green PCR Kit (Qiagen) as described previously [35 (link)].
Primer sequences are listed in Supplementary TableS2 .
Total cellular RNA was extracted using the RNeasy Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. One-step reverse transcription and real-time PCR were performed with a Quantstudio 5 (Thermo Fisher Scientific) using QuantiTect SYBR Green PCR Kit (Qiagen) as described previously [35 (link)].
Primer sequences are listed in Supplementary Table
10
ChIP Assay for ER Stress Markers
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