Cellytic mt buffer

Manufactured by Merck Group

Sourced in United States

CelLytic MT buffer is a lysis buffer designed for the extraction of proteins from mammalian cells. It is a non-ionic detergent-based buffer that efficiently solubilizes cell membranes and releases cellular proteins for further analysis.

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Lab products found in correlation

Complete mini protease inhibitor, by Roche (3 mentions) β actin, by Cell Signaling Technology (2 mentions) Nitrocellulose membrane, by Bio-Rad (2 mentions) Coomassie blue assay, by Thermo Fisher Scientific (2 mentions) Westernbreeze immunodetection system, by Thermo Fisher Scientific (2 mentions) Citrate synthase activity assay, by Merck Group (1 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Infinite m200 pro, by Tecan (1 mentions) Bca kit, by Thermo Fisher Scientific (1 mentions) Ecl reagent, by Merck Group (1 mentions) Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions) Protein assay dye reagent, by Bio-Rad (1 mentions) Odyssey infrared imager, by LI COR (1 mentions) Polyvinylidene difluoride membrane, by Merck Group (1 mentions) Sds page, by Bio-Rad (1 mentions) Ecl reagent, by Thermo Fisher Scientific (1 mentions) Immobilon fl pvdf membrane, by Merck Group (1 mentions) Odyssey blocking buffer pbs, by LI COR (1 mentions) Precast sds page gel, by Bio-Rad (1 mentions)

Close spelling variants of this product

CelLytic MT (44 citations) CelLytic buffer (36 citations) CelLytic MT lysis buffer (5 citations) CelLytic MT cell lysis buffer (4 citations) CelLytic MT Mammalian Tissue Lysis/Extraction buffer (3 citations)

15 protocols using cellytic mt buffer

Quantification of Citrate Synthase Activity in Kidney Tissue

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Kidney samples were washed twice in 0.9% (w/v) sodium chloride solution and homogenized in a CelLytic MT buffer (C3228; Sigma-Aldrich, St. Louis, MO, USA) supplemented with protease inhibitor cocktail (P8340; Sigma-Aldrich), and tissue disruption was completed by using a blunt-ended needle and a syringe. The lysates were centrifuged 16,000× g for 10 min at 4 °C, and supernatants were collected. The total protein concentration was determined using a Pierce™ BCA Protein Assay Kit (23227; ThermoFisher, Waltham, MA, USA). Ten micrograms of proteins was analysed by a citrate synthase activity assay (CS072; Sigma-Aldrich) according to the manufacturer’s protocol. The activity of citrate synthase was assessed by using the multimode microplate reader TECAN Infinite M200^® PRO (Tecan Group Ltd., Mannedorf, Switzerland) at 412 nm under a controlled temperature with a kinetic program.

Locatelli M., Macconi D., Corna D., Cerullo D., Rottoli D., Remuzzi G., Benigni A, & Zoja C. (2022). Sirtuin 3 Deficiency Aggravates Kidney Disease in Response to High-Fat Diet through Lipotoxicity-Induced Mitochondrial Damage. International Journal of Molecular Sciences, 23(15), 8345.

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Protein Extraction and Quantification from Transfected HUVECs

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Protein was extracted from TLR9 siRNA or scrambled RNA transfected HUVECs by Cellytic MT buffer (Sigma) according to the manufacture’s protocol. The concentration of protein was determined using BCA kit (Thermo Scientific). Membranes were incubated with HRP-conjugated secondary antibody (Cell signalling, MA, USA). The signals were developed with ECL reagent (Sigma) and captured by an electronic imaging system (Konica Minolta). β-actin (Cell signalling) was used as house-keeping control. Each samples was loaded as 10 μg protein in 40 μL. The quantification was achieved by normalizing each sample to its β-actin expression.

Wu J., Su W., Powner M.B., Liu J., Copland D.A., Fruttiger M., Madeddu P., Dick A.D, & Liu L. (2016). Pleiotropic action of CpG-ODN on endothelium and macrophages attenuates angiogenesis through distinct pathways. Scientific Reports, 6, 31873.

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Western Blot Analysis of Dio3 in Gill Tissue

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Gills were lysed using CelLytic MT buffer (Sigma-Aldrich, St. Louis, MO) with protease inhibitors (Calbiochem, Darmstadt, Germany), sonicated in ice bath, centrifuged at 14,000 × g for 15 min, 4°C, and supernatant was collected. Supernatant protein concentration was determined by the colorimetric Bradford assay using the Bio-Rad protein assay dye reagent (Bio-Rad, Hercules, CA). Samples consisting of 30 µg of total protein were separated by 4–20% SDS-PAGE (Bio-Rad), transferred to polyvinylidene difluoride membranes (Millipore) and incubated with specific antibody for Dio3 (Novus Biologicals) or β-actin (Cell Signaling) for 1 hour at room temperature, under agitation. Membranes were washed with PBS containing 0.01% Tween-20, incubated with secondary antibodies coupled to infrared fluorophores (Li-Cor Biosciences) for 30 min at room temperature, and blots were visualized using the Odyssey Infrared Imager (Li-Cor Biosciences). The expression of other selenoproteins was not measured due to the lack of specificity of commercially available antibodies.

Seale L.A., Gilman C.L., Moorman B.P., Berry M.J., Grau E.G, & Seale A.P. (2014). Effects of acclimation salinity on the expression of selenoproteins in the tilapia, Oreochromis mossambicus. Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements (GMS), 28(3), 284-292.

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Protein Extraction and Western Blot Analysis

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Cells were collected, and the whole cell lysates were prepared by sonication in CelLytic MT buffer from Sigma-Aldrich Co. Samples consisting of 50 µg of protein were loaded on a denaturing 4%–20% precast SDS-PAGE gel (Bio-Rad Laboratories Inc., Hercules, CA, USA) and then transferred to immobilon-FL PVDF membranes (EMD Millipore, Billerica, MA, USA) by electrophoresis. The membranes were blocked in Odyssey Blocking Buffer (PBS) (LI-COR) at room temperature for 1 hour and then incubated with specific primary antibodies at 4°C overnight. Blots were incubated with species-specific secondary antibodies at room temperature for 1 hour the next day. The signals were detected by ECL reagent (Thermo Fisher Scientific) and analyzed by ImageJ software (LOCI, University of Wisconsin, USA).

Zhang M., Jiang X., Qu M., Gu H., Sha Q, & Hua F. (2018). Salubrinal abrogates palmitate-induced leptin resistance and endoplasmic reticulum stress via nuclear factor kappa-light-chain-enhancer of activated B cell pathway in mHypoE-44 hypothalamic neurons. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 11, 893-899.

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Tissue Extraction and CrAT Activity Assay

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Tissues were ground into powder and processed in CelLytic MT buffer (Sigma Chemicals, St. Louis, MO) by freeze fracturing three times and sonication at five times one second pulses on setting five. CrAT activity was assessed according to (Seiler et al., 2014 (link)).

Seiler S.E., Koves T.R., Gooding J.R., Wong K.E., Stevens R.S., Ilkayeva O.R., Wittmann A.H., DeBalsi K.L., Davies M.N., Lindeboom L., Schrauwen P., Schrauwen-Hinderling V.B, & Muoio D.M. (2015). Carnitine Acetyltransferase Mitigates Metabolic Inertia and Muscle Fatigue During Exercise. Cell metabolism, 22(1), 65-76.

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Protein Extraction and Western Blot Analysis

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After harvest, 2x10⁶ cells were washed twice with PBS and lysed in CelLytic MT buffer (Sigma-Aldrich) supplemented with Mini Complete protease inhibitor (Roche), 1% phosphatase inhibitor cocktail (Calbiochem), and 1mM PMSF. After 10 min incubation on ice and centrifugation at 11,000g for 10 min at 4°C, the protein concentration was determined by BCA Assay (Thermo Scientific). Protein samples were loaded on 10% Bis-Tris gels and transferred on a PVDF membrane (BioRad). For protein detection of MTSS1 or GAPDH primary antibodies from Cell Signaling (#4386, clone N747, 1:1000 dilution) or Santa Cruz (sc-32233, clone 6C5, 1:2000 dilution) were used followed by a secondary goat anti-rabbit HRP conjugated antibody (Dako, 1:10000) and light emission analysis using Fusion SL system (PeqLab).

Schemionek M., Kharabi Masouleh B., Klaile Y., Krug U., Hebestreit K., Schubert C., Dugas M., Büchner T., Wörmann B., Hiddemann W., Berdel W.E., Brümmendorf T.H., Müller-Tidow C, & Koschmieder S. (2015). Identification of the Adapter Molecule MTSS1 as a Potential Oncogene-Specific Tumor Suppressor in Acute Myeloid Leukemia. PLoS ONE, 10(5), e0125783.

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Analyzing Tau Phosphorylation in DYRK1A Mice

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DYRK1A transgenic mice with three copies of the human DYRK1A gene were maintained as described previously (Ahn et al., 2006 (link)). Experiments were performed in accordance with the guidelines under the approval of the Institutional Review Committee for Animal Care and Use, KRIBB, Daejeon, Korea. The 8- to 10-week-old C57BL/6 mice were administered with DMSO or CX-4945 (75 mg/kg of body weight) orally in PBS solution, and the hippocampus was dissected after 30 min. The hippocampal lysates were prepared by using a Digital Sonifier Cell Disruptor instrument (BRANSON) in CelLytic MT buffer (catalog # C3228, Sigma-Aldrich) containing protease inhibitor cocktail set III (catalog # 535140-1ML, Calbiochem). The protein concentration was determined using the Bradford protein assay (Bio-Rad). Proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against phosphorylated Tau and Tau using an LAS4000 image analyzer (Fujifilm, Tokyo, Japan).

Kim H., Lee K.S., Kim A.K., Choi M., Choi K., Kang M., Chi S.W., Lee M.S., Lee J.S., Lee S.Y., Song W.J., Yu K, & Cho S. (2016). A chemical with proven clinical safety rescues Down-syndrome-related phenotypes in through DYRK1A inhibition. Disease Models & Mechanisms, 9(8), 839-848.

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Protein Expression Analysis by Western Blot

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Methods have been described previously.15 (link)–17 (link) In brief, after each treatment, whole-cell lysates were prepared by sonication in Cellytic MT buffer (Sigma-Aldrich) with protease/phosphatase inhibitors (Cell Signaling Technology) and cleared by centrifugation. Samples consisting of 40 µg proteins were resolved on a denaturing 4%–20% SDS-PAGE gel (Bio-Rad) and transferred to polyvinylidene fluoride membranes by electroblotting. Membranes were then blocked in PBS blocking buffer (Li-Cor) for 1 hour and incubated with specific primary antibodies at 4°C overnight. Blots were then incubated with species-specific secondary antibodies (Cell Signaling Technology). Signals were detected by a chemiluminescence reagent and analyzed with ImageJ software.

Gong T., Yu Y., Yang B., Lin M., Huang J.W., Cheng B, & Ji C. (2018). Celecoxib suppresses cutaneous squamous-cell carcinoma cell migration via inhibition of SDF1-induced endocytosis of CXCR4. OncoTargets and therapy, 11, 8063-8071.

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Protein Expression Analysis by Western Blotting

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After different treatments, cells were lysed by sonication in CelLytic MT buffer (Sigma, St. Louis, MO, USA) and proteins were extracted. 50 μg protein samples were separated on a denaturing 10% SDS-PAGE gel, and transferred to polyvinylidene fluoride membranes. The membrane was then blocked in PBST with 5% non-fat milk at room temperature for 1 h, followed by incubation with specific primary antibodies at 4 °C overnight. Blots were incubated with appropriate secondary antibodies at room temperature for 1 h the next day. The signals were detected by ECL detection kit. Blots were analyzed by ImageJ software.

Zhang Y., Chen C., Jiang Y., Wang S., Wu X, & Wang K. (2017). PPARγ coactivator-1α (PGC-1α) protects neuroblastoma cells against amyloid-beta (Aβ) induced cell death and neuroinflammation via NF-κB pathway. BMC Neuroscience, 18, 69.

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Measuring Placental LDH Activity

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Placental tissue (50 mg) was homogenized in 500 μl ice‐cold CelLytic MT Buffer (Sigma‐Aldrich). LDH activity was assessed using the LDH Activity Assay (ab102526, Abcam). Protein concentrations were determined as described above and 1 μg protein was loaded per reaction in duplicate. Assays were performed at 37℃ and A₄₅₀ was measured every 5 min for 40 min. The Δ A₄₅₀ was calculated for 20 min of the linear reaction between 20‐ and 40‐min time points and is proportional to the NADH concentration produced by the LDH enzymatic reaction converting lactate into pyruvate. Results were normalized to the amount of protein loaded in the reaction.

Jones A.K., Rozance P.J., Brown L.D., Lorca R.A., Julian C.G., Moore L.G., Limesand S.W, & Wesolowski S.R. (2021). Uteroplacental nutrient flux and evidence for metabolic reprogramming during sustained hypoxemia. Physiological Reports, 9(18), e15033.

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