Anti mecp2

Manufactured by Merck Group

Sourced in United States, Canada, France

Anti-MeCP2 is a laboratory equipment product manufactured by Merck Group. It serves as a tool for the detection and quantification of MeCP2 protein, which is a key regulator of gene expression. The product functions by providing a reliable and specific method for measuring MeCP2 levels in various biological samples.

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Lab products found in correlation

Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Lumi light western blotting substrate kit, by Roche (1 mentions) Anti β actin, by Santa Cruz Biotechnology (1 mentions) Chemidoctm mp imaging system, by Bio-Rad (1 mentions) Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions) 0.2 μm nitrocellulose membrane, by Bio-Rad (1 mentions) Image studio lite software, by LI COR (1 mentions) Irdye 800cw donkey anti mouse igg, by LI COR (1 mentions) Anti γ tubulin, by Merck Group (1 mentions) Complete edta free cocktail, by Roche (1 mentions) Odyssey infrared imager, by LI COR (1 mentions) Homogenizer, by PRO Scientific (1 mentions) Protease inhibitor pmsf, by Beyotime (1 mentions) Imager 600, by Cytiva (1 mentions) Mouse anti actin, by Merck Group (1 mentions) Ripa buffer, by Beyotime (1 mentions) Anti neun, by Merck Group (1 mentions) Light chain specific secondary antibody, by Jackson ImmunoResearch (1 mentions) Anti ncor1, by Cell Signaling Technology (1 mentions) Anti srebp2, by Cell Signaling Technology (1 mentions)

8 protocols using anti mecp2

Immunoblotting for MeCP2 and β-catenin

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Cell lysates were prepared by washing cells with ice-cold PBS twice, and then lysing on ice for 5 min with RIPA. Samples were assayed for protein concentration using Coomassie (Bradford) Protein Assay Kit (Thermo Scientific), and then mixed with 6x SDS sample buffer before resolving by 6%-10% SDS-PAGE, transferring onto PVDF membrane, and immunoblotting. For immunoblotting, membrane was incubated with anti-MeCP2 (1:1000, Abcam, Sigma Chemical Co.), anti-non-phosphorylated (S33/37/T41) β-catenin (1:1000, Cell Signaling Technology), and anti-β-actin (1:5000, Santa Cruz Biotechnology) antibodies in 5% non-fat-milk/1x TBS containing 0.1% Tween20 overnight at 4°C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). Proteins were detected by a chemiluminescent method using Lumi-Light Western Blotting Substrate kit (Roche), and/or SuperSignal West Femto kit (Pierce). Densimetry of detected bands for protein of interest was performed by using Bio-Rad ChemiDoc^TM MP Imaging System and the data were standardized by β-actin density.

Kweon S.M., Chi F., Higashiyama R., Lai K, & Tsukamoto H. (2016). Wnt Pathway Stabilizes MeCP2 Protein to Repress PPAR-γ in Activation of Hepatic Stellate Cells. PLoS ONE, 11(5), e0156111.

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Western Blot Analysis of MeCP2 Protein

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One frozen half brain was homogenized in ice-cold NE1 buffer [20 mm Hepes pH 7.9, 10 mm KCl, 1 mm MgCl₂, 0.1% Triton X-100, 20% glycerol, 0.5 mm DTT, protease inhibitor (complete EDTA free cocktail, Roche)] before adding 750 units of benzonase for 15 min at room temperature then an equal volume of 2× SDS loading buffer (0.125 M Tris pH 6.8, 20% glycerol, 4% SDS, 0.25% bromophenol blue, 20 mm DTT, 0.3 M β-mercaptoethanol). Samples were boiled for 3 min before loading equal volumes on a 4–12% Run Blue SDS precast gel (Expedeon). Gels were transferred to a 0.2-μm nitrocellulose membrane (Bio-Rad) and then blocked for 1 h (5% milk, 1× TBS, 0.1% Tween) before applying anti-Mecp2 1:1000 (Sigma M6818) or anti-γ-tubulin 1:3000 (Sigma T5326) overnight at 4°C, followed by IRDye 800CW donkey anti-mouse IgG 1:10 000 (LiCor) for 2 h at RT after washing. Membranes were imaged using the Odyssey Infrared Imager (LiCor) and quantified using Image Studio Lite Software (LiCor).

Brown K., Selfridge J., Lagger S., Connelly J., De Sousa D., Kerr A., Webb S., Guy J., Merusi C., Koerner M.V, & Bird A. (2015). The molecular basis of variable phenotypic severity among common missense mutations causing Rett syndrome. Human Molecular Genetics, 25(3), 558-570.

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Western Blot Analysis of MeCP2 in Brain

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Brains were dissected into ice-cold PBS, the hippocampus and parietal cortex were rapidly dissected out. Tissue samples were lysed with 500 μl ice-cold RIPA buffer (Beyotime Biotechnology) containing 1 mM protease inhibitor PMSF (Beyotime Biotechnology) and PIC (Sigma). Cell lysates were homogenized by a homogenizer (PRO Scientific, Inc.) and the protein concentration was determined using a BCA Protein Assay Kit (Thermofisher, 23227). Protein lysates were dissolved in SDS-PAGE protein loading buffer and heated at 95 ^oC for 10 min. Samples were separated on 10% SDS-PAGE gels (Epizyme, Lnc) and transferred onto PVDF membranes (Immobillon). After blocking with TBST solution containing 5% skimmed milk for 1 h at room temperature, membranes were incubated with primary antibodies (rabbit anti-MeCP2, 1:2000; cell signaling, 3456S; mouse anti-Actin, 1:5000; Sigma, A4700–2ML) followed by HRP-conjugated secondary anti-rabbit and anti-mouse antibodies (1:5000; Vector lab). Blots were imaged with Amersham Imager 600 and quantified using the Image J Fiji software.

Lu Z., Liu Z., Mao W., Wang X., Zheng X., Chen S., Cao B., Huang S., Zhang X., Zhou T., Zhang Y., Huang X., Sun Q, & Li J.D. (2020). Locus-specific DNA methylation of Mecp2 promoter leads to autism-like phenotypes in mice. Cell Death & Disease, 11(2), 85.

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Immunofluorescent Staining of Hippocampal Neurons

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Mice (1 month old) were deeply anesthetized with chloral hydrate (4%; 1 ml/100 g body weight, i.p.) and perfused intracardially with 4% paraformaldehyde. Immunofluorescent staining was carried out on free-floating sections as described in (Menna et al., 2013 (link)). Free-floating sections at the level of dorsal hippocampus were processed with the specific antibodies as indicated, followed by incubation with the secondary antibodies, counterstained with DAPI and mounted in Fluorsave (Calbiochem, San Diego, CA, USA).
Primary antibodies: anti-vGLUT-1 (guinea pig polyclonal antibody, 1:1000; No. 135 304 Synaptic System), anti-MeCP2 (rabbit polyclonal, 1:200; M9317 Sigma), anti-NeuN (mouse monoclonal, 1:500; MAB377 Millipore). Sections were examined by means of a Zeiss LSM 510 META confocal microscope (Leica Microsystems, Germany). Images were acquired in the stratum pyramidale or stratum radiatum of the CA1 subfield of the hippocampus (as indicated) using the x40 oil immersion lens with an additional electronic zoom factor of up to 3 (voxel sizes of 0.10 × 0.10×1 μm) maintaining the parameters of acquisition (laser power, pinhole, gain, offset) constant among groups.

Tomasoni R., Morini R., Lopez-Atalaya J.P., Corradini I., Canzi A., Rasile M., Mantovani C., Pozzi D., Garlanda C., Mantovani A., Menna E., Barco A, & Matteoli M. (2017). Lack of IL-1R8 in neurons causes hyperactivation of IL-1 receptor pathway and induces MECP2-dependent synaptic defects. eLife, 6, e21735.

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Western Blot Analysis of Protein Targets

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The following antibodies were used for western blotting: anti-MeCP2 (Millipore 07-013) at a concentration of 1:700 in 3% Milk-PBS; anti-SREBP2 (ab30682) at a concentration of 1:400 in 5% Milk-TBST; anti-GAPDH (Cell Signaling 5174) at a concentration of 1:8000 in 5% Milk-TBST; anti-NCoR1 (sc-1609) at a concentration of 1:450 in 1% casein-TBST; anti-TBLR1 at a concentration of 1:1200 in 1% casein-TBST. For co-IP, a light-chain specific secondary antibody (Jackson ImmunoResearch) was used to avoid heavy chain detection at 50kDa. Quantification of band intensity was achieved using ImageJ (NIH) and normalized to GAPDH.

Kyle S.M., Saha P.K., Brown H.M., Chan L.C, & Justice M.J. (2016). MeCP2 co-ordinates liver lipid metabolism with the NCoR1/HDAC3 corepressor complex. Human Molecular Genetics, 25(14), 3029-3041.

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Chromatin Immunoprecipitation Assay for NRF2

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The chromatin immunoprecipitation (ChIP) assays were performed using the MAGnify™ Chromatin Immunoprecipitation System (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Briefly, LNCaP cells treated with DMSO or 5-Aza/TSA for 7 days were washed with PBS and trypsinized. The trypsinized cells were then washed with PBS, and the chromatin from the cells (100,000 cells were used per IP) was cross-linked with 1% formaldehyde for 10 min at room temperature and sonicated to generate ~200- to 500-bp DNA fragments in lysis buffer. The proteins were then immunoprecipitated with 6 to 10 µl of anti-Pol II, anti-MBD2, anti-MeCP2, anti-trimethyl-histone H3-Lys9 (H3K9me3) and anti-H3Ac antibodies (Millipore, MA) or mouse IgG to capture DNA-bound protein complexes. The enrichment of the eluted DNA was quantified by comparison with the input lysate by qPCR (29 ) using designed primers covering a 93-bp fragment that is 29 bp upstream of the first CpG site in the NRF2 promoter (Supplemental Table S1).

Khor T.O., Fuentes F., Shu L., Paredes-Gonzalez X., Yang A.Y., Liu Y., Smiraglia D.J., Yegnasubramanian S., Nelson W.G, & Kong A.N. (2014). Epigenetic DNA Methylation of Anti-oxidative Stress Regulator NRF2 in Human Prostate Cancer. Cancer prevention research (Philadelphia, Pa.), 7(12), 1186-1197.

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Pharmacological Evaluation of Diazepam and Imidazenil

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Adult male Sprague Dawley rats (Harlan, Indianapolis) were received and after 1 week of acclimatization they were housed individually, maintained on a 12/12‐h light‐dark cycle with standard food and water made available ad libitum. All experiments were carried out in accordance with the National Institute of Health Guidelines for the Care and Use Laboratory Animals and approved by the animal committee at the University of Illinois at Chicago.
Diazepam and imidazenil were obtained from Hoffman‐La Roche (Nutley, NJ); TRIzol reagent was obtained from Life Technologies (Life Technologies Corporation, USA); Qiagen RNeasy Kit from Qiagen (Valencia, CA, USA); Fermentas Maxima SYBR Green/ROX qPCR Master Mix obtained from Fermentas (Fermentas International Inc., Canada); Novex 4%‐12% Tris‐Glycine gels obtained from Invitrogen (Carlsbad, CA); HRP‐conjugated secondary anti‐mouse or anti‐rabbit antibodies from GE Healthcare (Arlington Heights, IL); anti‐GAPDH, Immobilon Western Chemiluminescent HRP Substrate, anti‐H3K9me2, anti‐MeCP2, anti‐H3, and anti‐acetyl‐H3 from Millipore (Billerica, MA).

Auta J., Gatta E., Davis J.M., Pandey S.C, & Guidotti A. (2018). Potential role for histone deacetylation in chronic diazepam‐induced downregulation of α1‐GABAA receptor subunit expression. Pharmacology Research & Perspectives, 6(4), e00416.

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Comprehensive Neurological Marker Profiling

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Primary antibodies were as follows: anti-βIII-Tubulin (mouse monoclonal, Sigma, France), anti-βIII-Tubulin (rabbit polyclonal, Abcam, France), anti-actin (mouse monoclonal, Sigma, France), anti-ApoE (goat polyclonal, Millipore, France), anti-Cleaved caspase-3 (rabbit polyclonal, Cell Signaling, France), anti-GABA (rabbit polyclonal, Sigma, France), anti-GFAP (rabbit polyclonal, DAKO, France), anti-vGLUT1 (rabbit polyclonal, Abcam, France) anti-glutamate transporter neuronal (EAAC1) (goat polyclonal, Millipore, France), anti-vGLUT1 and anti-vGLUT2 (rabbit polyclonal, Synaptic Systems, Germany), anti-HuC/D (mouse monoclonal, Molecular Probes, Life Technologies, France), anti-MAP2 (mouse monoclonal, Sigma, France), anti-MeCP2 (rabbit, a generous gift from Dr E. Joly, Toulouse, France), anti-REST (rabbit polyclonal, Millipore, France), anti-SCG10 (rabbit polyclonal, a generous gift from Dr A. Sobel, Paris, France), anti-TH (rabbit polyclonal, Abcam, France), anti-SOX2 (rabbit polyclonal, Millipore, France) and anti-BDV-P and anti-BDV-X (rabbit polyclonal antibody).

Scordel C., Huttin A., Cochet-Bernoin M., Szelechowski M., Poulet A., Richardson J., Benchoua A., Gonzalez-Dunia D., Eloit M, & Coulpier M. (2015). Borna Disease Virus Phosphoprotein Impairs the Developmental Program Controlling Neurogenesis and Reduces Human GABAergic Neurogenesis. PLoS Pathogens, 11(4), e1004859.

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