Rabbit anti-Yap antibody (ab62752, Abcam) or rabbit anti-Rif1 antibody (Covalab, custom-made antibody), pre-immune serum or rabbit IgG (M7023, Sigma) were coupled overnight at 4 °C to protein A Sepharose beads (GE Healthcare). Coupled beads were washed three times in EB buffer (50 mM Hepes, pH 7.5, 50 mM KCl, 5 mM MgCl2). For Yap depletions, coupled beads were then incubated 1 hr at 4 °C in egg extracts (volume ratio 1:3). For Rif1 depletions, coupled beads were incubated 30 min at 4 °C in egg extracts (ratio 1:1) and egg extracts after a first round were re-incubated another 30 min at 4 °C with coupled beads.
M 7023
Manufactured by Merck Group
The M-7023 is a laboratory instrument designed for performing various analytical tasks. It is a versatile equipment that can be utilized in a wide range of scientific applications. The core function of the M-7023 is to provide accurate and reliable data measurements. The detailed specifications and intended use of this product are not available for an unbiased and factual description.
Automatically generated - may contain errors
Lab products found in correlation
Ab62752, by Abcam (1 mentions)
Protein a sepharose beads, by GE Healthcare (1 mentions)
Ab125096, by Abcam (1 mentions)
Ab2402, by Abcam (1 mentions)
Alexa antibodies, by Thermo Fisher Scientific (1 mentions)
Ab8895, by Abcam (1 mentions)
Ab8580, by Abcam (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
4 protocols using m 7023
1
Immunoprecipitation of Yap and Rif1
2
Immunofluorescence and Immunoprecipitation Protocols
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Primary antibodies used during immunofluorescence experiments are listed below. Rabbit polyclonal anti-TOPBP1 (1:500; Ab2402; ABCAM); rabbit polyclonal anti-53BP1 (1:500; Ab2402; NOVUS BIO); mouse monoclonal anti-phospho-H2A.X (1/500; CAT05-636; Millipore); rabbit polyclonal anti-phospho-H2A.X (1:250; 2577S; Cell Signaling); mouse monoclonal anti-GFP (1/500; 1184144600; Roche); mouse monoclonal anti-mCherry (1/250; AB125096; ABCAM); rabbit polyclonal anti-mCherry (1:250; 632496; Clontech); secondary antibodies used for detection were Alexa antibodies (In Vitrogen). The antibodies used during immunoprecipitation experiments are listed below. Mouse monoclonal anti-BrdU (1:40; 555627; BD); rabbit polyclonal anti-mouse (3.45 μl/sample; M-7023; Sigma).
3
Histone ChIP-qPCR: Profiling Epigenetic Marks
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Histone ChIP followed by quantitative PCR (qPCR) was carried out as previously described17 (link). Briefly, 50 μg of precleared cross-linked chromatin from TS cells was incubated overnight with 5 μg of antibody followed by 4 h with Sepharose A beads. Eluates were digested with Proteinase K (0.5 mg/ml) and Rnase A (0.1 mg/ml), and DNA was phenol-choloroform extracted and recovered by standard precipitation with ethanol. The following antibodies were used: anti-H3K4me1 (ab8895; Abcam), anti-H3K4me3 (ab8580; Abcam), anti IgG (m7023, Sigma). Supplementary Table S2 lists the primers used for ChIP. ChIP data are represented as the fraction of the differences in ct values for each specific primer set between histone mark and IgG divided by the IgG ct value.
4
Chromatin Immunoprecipitation (ChIP) Protocol
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Cross-linking was performed with formaldehyde (Merck) at a final concentration of 1% and terminated after five minutes by addition of glycine at a final concentration of 0.125 M. Cells were harvested with SDS buffer (50 mM Tris pH 8.1, 0.5% SDS, 100 mM NaCl, 5 mM EDTA) and resuspended in IP buffer after pelleting (2 parts of SDS buffer and 1 part Triton dilution buffer (100 mM Tris-HCl pH 8.6, 100 mM NaCl, 5 mM EDTA, pH 8.0, 0.2% NaN3, 5.0% Triton X-100)). Chromatin was sheered by sonication (HTU SONI 130, G. Heinemann) to generate DNA fragments with an average size of 500 bp. Preclearing and the incubation with AP4 antibody (AbD Serotec) or the respective IgG control (M-7023, Sigma) for 16 hours was performed as previously described [24 (link)]. Washing and reversal of cross-linking was performed as described [37 (link)]. Immunoprecipitated DNA was analyzed by qPCR and the enrichment was expressed as percentage of the input for each condition [37 (link)]. The sequences of oligonucleotides used as qChIP primers are listed in Table S1 .
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