Cells were resuspended in lysis buffer (1% SDS; 10mM EDTA; 50mM Tris-Hcl pH8, 1; 1mM PMSF; 10 μg.ml−1 aprotinine; 10 μg.mL−1 leupeptine; 10 μg.mL−1 pepstatine; 1mM Na3VO4 and 50mM NaF) and sonicated with Bioruptor apparatus from Diagenode. Protein concentration was measured using bicinchoninic acid (BCA protein assay, Pierce, Rockford, IL, USA). Fifty micrograms of proteins were loaded for each lane and separated by 10%, 12.5% or 15% SDS-PAGE, then electrotransfered to PVDF membranes. Western blot analysis was performed by standard techniques with ECL detection (Bio-Rad, Marne-la coquette, France).
Bioruptor apparatus
Manufactured by Diagenode
Sourced in United States
The Bioruptor apparatus is a device used for the fragmentation of biological samples, such as DNA, RNA, or proteins, through the application of ultrasonic waves. The device utilizes a water bath to ensure uniform and efficient sample processing. The core function of the Bioruptor is to provide a controlled and reproducible method for the mechanical disruption of samples.
Automatically generated - may contain errors
Lab products found in correlation
Magmedip kit, by Diagenode (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Histone h3, by Abcam (1 mentions)
Lipofectamine 3000, by Merck Group (1 mentions)
Abi 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Myc tag, by Abcam (1 mentions)
4 protocols using bioruptor apparatus
1
Protein Extraction and Western Blotting
2
MeDIP-Seq Protocol for DNA Methylation Analysis
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MeDIP experiments were performed using MagMEDIP kit from Diagenode (#C02010020), according to the manufacturer's instructions. Briefly, genomic DNA was extracted from 3 to 10 million cells and sonicated using the Bioruptor apparatus (Diagenode) to obtain fragments of 100–600 bp in length (10 min at ‘low' power with cycles of 15 s ON and 15 s OFF). For each reaction, 1 μg DNA was used. Positive (methylated DNA) and negative (unmethylated DNA) controls were added to the reaction, in order to normalize for the immunoprecipitation (IP) efficiency. IP using anti-5-methylcytosine antibody and magnetic beads was performed overnight at 4 °C. After washing and elution, immunoprecipitated DNA was measured by qPCR and reported to input DNA.
3
c-Fos Chromatin Immunoprecipitation in HEK 293T Cells
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Chromatin immunoprecipitation was performed using HEK 293T cells. Chromatin was sheared on ice by sonication using a Bioruptor apparatus (Diagenode, USA) for six 1-min cycles at a high intensity (200 W) and 20 s off. The size of the sheared chromatin was approximately 200-1,000 bp, as determined by agarose gel electrophoresis. After adding the anti-c-Fos antibody (Table 1 ), nonspecific binding was blocked by incubation with A/G agarose for 1 h at 4°C. Thereafter, ChIP was performed using a standard protocol. ChIP DNA was analyzed by qPCR using the TOPrealTM SYBR Green qPCR PreMIX (RT500S; Enzynomics) and the 7500 Real-Time PCR System (Applied Biosystems).
4
Chromatin Immunoprecipitation Sequencing in HEK293 Cells
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HEK2935xUAS cells were transfected twice with Lipofectamine 3000 with the appropriate constructs and fixed 24 h later with 1% (v/v) formaldehyde (Sigma) for 10 min at room temperature before quenching of the cross‐linker with 125 mM glycine. Preparation of chromatin was performed as described previously (Tardat et al, 2015 ). Chromatin was sheared with a Bioruptor apparatus (Diagenode) to obtain DNA fragments with size between 200 and 800 bp. The lysates were incubated overnight at 4°C with Dynabeads protein G (Invitrogen) coupled to the appropriate antibody: GAL4 DNA‐binding domain (Millipore, Cat#06‐262), Myc‐tag (Abcam, Cat#Ab9132), RNF2 (Active Motif, Cat#39663), Histone variant H3.3 (Millipore, 09‐838), and Histone H3 (Abcam, Cat#1791). Recovered DNA was purified using the MinElute PCR purification kit (Qiagen Cat#28006) following the manufacturer's instructions. For Sequencing, the libraries were prepared with the ChIP‐Seq NEB Ultra Kit (New England Biolabs Cat# E6200) and sequenced with the Illumina Hiseq (50 cycles and single‐end reads). Quantitative PCR analyses of immunoprecipitated DNA were performed in triplicate using a Fast SYBR Green Master Mix (Thermo Fisher Scientific Cat#4309155) on an ABI 7500 Fast Real‐Time PCR System (Applied Biosystems). The primer pairs used are indicated in Table EV3 .
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