ChIP assays were performed using the Pierce Magnetic ChIP Kit according to the manufacturer’s instructions70 (link). Briefly, cross-linking with 1% formaldehyde was carried out in RTECs or renal tissues, followed by quenching with glycine, cell harvesting, and DNA fragmentation by sonication. Lysates were precleared for 1 h with Protein A + G magnetic beads (EMD Millipore). Precleared lysates were then incubated with 5 μg of anti-SOX9 antibodies (Abcam, ab3697) overnight at 4 °C, followed by addition of Protein A + G magnetic beads and incubation for 4 h at 4 °C. Subsequently, the beads were repeatedly washed, followed by elution of the protein–DNA complexes, reversal of cross-links, and DNA purification. Standard qPCR analysis was then carried out using primers spanning the promoters of target genes. The sequences of primers are shown in Supplementary Table 2 .
Protein a g magnetic beads
Manufactured by Abcam
Sourced in United States
Protein A/G magnetic beads are a versatile tool used in various immunoassays and biomolecular purification applications. They provide a convenient and efficient way to capture and isolate target proteins or other biomolecules from complex samples. The beads are coated with recombinant Protein A and Protein G, which have a high affinity for the Fc region of antibodies, enabling the rapid and selective isolation of antibodies or antibody-bound complexes.
Automatically generated - may contain errors
Lab products found in correlation
Protein a g magnetic bead, by Thermo Fisher Scientific (3 mentions)
Ab3697, by Abcam (1 mentions)
Protein a g magnetic beads, by Merck Group (1 mentions)
Ne per nuclear and cytoplasmic extraction reagents kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Sds sample buffer, by Boston BioProducts (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti p105 p50, by Cell Signaling Technology (1 mentions)
Anti relb, by Abcam (1 mentions)
H3k27me3, by Abcam (1 mentions)
Washing buffer, by Thermo Fisher Scientific (1 mentions)
Magnetic separation device, by Thermo Fisher Scientific (1 mentions)
Ab196026, by Abcam (1 mentions)
Ab275378, by Abcam (1 mentions)
Ab181053, by Abcam (1 mentions)
Bs 6982r, by Bioss Antibodies (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Magna rip immunoprecipitation kit, by Merck Group (1 mentions)
7 protocols using protein a g magnetic beads
1
ChIP Assay for SOX9 Transcriptional Regulation
2
Immunoprecipitation of Nuclear Proteins
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After 40h incubation with indicated stimuli, mDCs were washed with ice-cold PBS and lysed using NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific, 78833). Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, 78442) was used according to the manufacturer’s instructions. Protein concentrations of cytoplasmic and nuclear extracts were measured using Pierce BCA Protein Assay Kit (Thermo Scientific, 23227). Nuclear proteins of mDCs (25 μg/group) were pre-cleared by incubation with Protein A/G Magnetic Beads (Thermo Scientific, 88802) for 1h with continuous rotation at 4°C. After removal of beads, nuclear proteins were incubated with anti-p105/p50 (Cell Signaling Technology, 13586S) or anti-RelB (Abcam, ab33917) and Protein A/G Magnetic Beads overnight with continuous rotation at 4°C. Beads were then harvested, followed by extensive wash. Immunoprecipitants were eluted and denatured by heating with SDS-Sample Buffer (Boston BioProducts, BP-111R) at 90°C for 5 minutes. Proteins were then separated by electrophoresis in SDS/PAGE and transferred to PVDF membrane (Bio-Rad, 1620177) for immunoblotting.
3
Chromatin Immunoprecipitation (ChIP) Assay
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HL‐60 and NB4 cells (~5 × 106) were washed and incubated in 1% formaldehyde solution on ice for crosslinking. Then, the formaldehyde solution was removed, and cells were washed, detached and suspended in cell lysis buffer for 30 min on ice. Cell lysates were collected and sonicated to obtain DNA fragments (~500 bp). Rabbit anti‐human EZH2 (5 μg, Abcam), H3K27me3 (5 μg, Abcam) and EBF3 (5 μg, Thermo Fisher Scientific) and isotype control IgG (5 μg) were added to the DNA fragments and incubated for 16 h at 4°C. Subsequently, Protein A/G Magnetic Beads (Abcam) were mixed with samples and incubated for 2 h. DNA was eluted and subjected to qPCR. Primers are shown in Table S1 .
4
Magna RIP Immunoprecipitation Protocol
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The RIP experiment was performed using a Magna RIP immunoprecipitation kit (Merck Millibo, Billerica, Massachusetts, USA). After the PDAC cells were lysed, the supernatant was collected and antibody was added and incubated overnight. Next, protein A/G magnetic beads (Abcam Inc., Cambridge, MA, USA) were added and incubated again for 1 h. Finally, the results were verified by RT-qPCR 32 (link).
5
Immunoprecipitation of G-CSF and EP300
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KG1a cells were lysed by the non-denaturing lysis buffer. Next, the supernatant of cell lysis was pre-cleaned with protein A/G magnetic beads (Thermo Fisher Scientific) for 2 h at 4 °C. Subsequently, about 300 μg of protein were incubated with 1μg G-CSF antibody (#ab181053, Abcam) or EP300 antibody (#ab275378, Abcam) and 25 microliters of protein A/G magnetic beads for immunoprecipitation at 4 °C overnight. Following the incubation with antibody and protein A/G magnetic beads, protein A/G magnetic beads were collected using magnetic separation device (Thermo Fisher Scientific), and precipitated complexes were cleansed by washing buffer (Thermo Fisher Scientific). Finally, bound proteins were analyzed by WB using anti-ELANE (1:500, # bs-6982R, Bioss) or anti-TPO (1:500, # ab196026, Abcam). Rabbit IgG was used for negative control.
6
Immunoprecipitation of CSB Protein
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Whole cell extracts were prepared from untreated HeLa cells, or where indicated, at 0.5 or 2 h after 6 μM trioxsalen/UVA treatment. In brief, cells were lysed in 20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton-X-100, 1 mM ethylenediaminetetraacetic acid (EDTA) and complete protease inhibitor cocktail (Roche, Mannheim, Germany) with sonication, and insoluble material was removed by centrifugation at 14 000 × g for 10 min at 4°C. Prior to α-CSB immunoprecipitation, the soluble whole cell extract was pre-treated with protein A/G magnetic beads (Thermo Fisher Scientific) at 4°C for 2 h to remove non-specific protein binders. Extracts were then incubated with mouse α-CSB antibody (ab66598; Abcam, Cambridge, MA, USA) for 12 h, and the immunocomplexes were captured by protein A/G magnetic beads for 2 h at 4°C. The bead-bound material was washed five times, suspended in 2× SDS-PAGE loading dye and incubated at 95°C for 5 min. Proteins were resolved on a polyacrylamide gel and detected by western blotting.
7
LINC00158 Enrichment via RIP
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RIP experiment was carried out using Magna RIP Immunoprecipitation Kit (Millipore, Billerica, MA, USA). Brie y, cells were lysed using RIP buffer and incubated with ProteinA/G magnetic beads bounded with primary antibodies against Ago2 or IgG (Abcam, Cambridge, MA, USA). Enrichment of LINC00158 was assessed by qRT-PCR.
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