Cellytic mt lysis buffer

Manufactured by Merck Group

CelLytic MT lysis buffer is a non-denaturing, detergent-based buffer designed for the extraction and solubilization of proteins from mammalian cells. It is optimized to maintain the native structure and function of proteins during the lysis process.

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Lab products found in correlation

Gs 700 imaging densitometer, by Bio-Rad (1 mentions) Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Memcode staining, by Thermo Fisher Scientific (1 mentions) Bca kit, by Merck Group (1 mentions) Cryoprep instrument, by Covaris (1 mentions) Tissuelyser lt, by Qiagen (1 mentions) Protease inhibitor, by Merck Group (1 mentions) Trans blot sd, by Bio-Rad (1 mentions) Bcip nbt, by Merck Group (1 mentions)

5 protocols using cellytic mt lysis buffer

Protein Expression Analysis in Tumor Samples

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Cells were harvested and lysed in CelLytic^MT lysis buffer (Sigma-Aldrich) after either 24 or 48 h of treatment, and total protein was separated by SDS-PAGE and transferred to the membranes and immunostained using antibodies specific to the indicated proteins. For in vivo pharmacodynamic studies, end of study flash-frozen tumors (4 h post-last dose) were fractured using a cryoPREP instrument (Covaris), and pulverized tissue was lysed in CelLytic^MT lysis buffer (Sigma-Aldrich). Total protein was analyzed by Western blot analysis as described above. Protein expression was analyzed using standard practice and antibodies as follows: ERα, PR, E2F1, CCNE1, CCNE2, CCND1, total Rb, phospho-Rb S807/811, CDK2, CDK4, CDK6, Actin (Cell Signaling Technologies, Catalog #13258, #3153, #3742, #20808, #4132, #2978, #9309, #8516, #2546, #12790, #13331, #4970, respectively), phospho-p107, p107, phospho-p130, p130 (Abcam: ab111348, ab76255, ab168458, ab6545, respectively), GREB1 (Millipore, MAB562), and Vinculin (Sigma-Aldrich, #v9131). Protein expression was quantified using the AzureSpot software and normalized to the expression of the vinculin protein.

Patel H.K., Tao N., Lee K.M., Huerta M., Arlt H., Mullarkey T., Troy S., Arteaga C.L, & Bihani T. (2019). Elacestrant (RAD1901) exhibits anti-tumor activity in multiple ER+ breast cancer models resistant to CDK4/6 inhibitors. Breast Cancer Research : BCR, 21, 146.

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Quantitative Western Blot Analysis of Mn-SOD

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Cell lysates were subjected to western blot analysis, as described previously (4 (link)). EPCs were lysed using CelLytic MT lysis buffer (Sigma-Aldrich) with protease inhibitor cocktail (100 µl protease inhibitor/10 ml lysis buffer; Sigma-Aldrich). The protein content was assayed by bicinchoninic acid assay (Pierce Biotechnology, Inc., Rockford, IL, USA). Proteins (20 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membrane was incubated with a 1:1,000 dilution of primary antibody targeting Mn-SOD or GAPDH, followed by a 1:5,000 dilution of horseradish peroxidase-conjugated secondary antibody. Protein bands were visualized by electrochemiluminescence (ECL Prime Western Blotting Detection Reagent; cat no. RPN2232; GE Healthcare Life Sciences, Shanghai, China). The intensity (area × density) of the individual bands on the western blots was measured by densitometry (model GS-700 Imaging Densitometer; Bio-Rad Laboratories, Inc.). The background was subtracted from the calculated intensity.

JIN P., LI T., LI X., SHEN X, & ZHAO Y. (2016). Suppression of oxidative stress in endothelial progenitor cells promotes angiogenesis and improves cardiac function following myocardial infarction in diabetic mice. Experimental and Therapeutic Medicine, 11(6), 2163-2170.

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Protein Expression Analysis Protocol

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Protein was isolated using CelLytic MT lysis buffer (Sigma Chemicals, St. Louis, MO) and quantified by using a bicinchoninic acid (BCA) kit (Sigma Chemicals, St. Louis, MO). Protein (50μg from cell/tissue lysates) was separated by SDS-PAGE, transferred to nitrocellulose, and incubated with antibodies prepared with fish gelatin blocking buffer (0.5% fish gelatin, 1X PBS, 1 mg/ml casein and 15 mM NaN₃). IgG-congugated AlexFluor fluorescent secondary antibodies (Invitrogen) were used with the Li-COR (Li-COR Biosciences) to visualize bands. Protein expression was normalized to total protein as determined by MemCode staining (Thermo Scientific). The primary CrAT antibody was a generous gift from the laboratory of Dr. Fausto Hegardt.

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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Cytokine Profiling of Mouse Colon Tissue

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Mouse colon tissues were suspended in 400 uL CelLytic MT lysis buffer (Sigma-Aldrich, St. Louis, MO) with protease inhibitors (EMD Millipore, Billerica, MA) and homogenized at 50Hz for 2 minutes with a stainless steel bead using a TissueLyser LT (Qiagen, Germantown, MD). Homogenates were incubated on ice for 10 minutes prior to a 7 minute, 14,000 × g centrifugation at 4°C. Total protein concentration of the resulting supernatants was determined by colorimetric protein assay and aliquots were stored at -80°C. Cytokine concentrations (G-CSF, GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17A, IP-10, KC, MCP-1, MIP-1α, MIP-1β, MIP-2, RANTES, TNFα) were determined by multiplex analysis using a Milliplex MAP Mouse Magnetic Bead Panel (EMD Millipore, Billerica, MA). IL-13 was not detected by Luminex assay and an ELISA was used (eBioscience, San Diego, CA). Tissue lysate concentrations were normalized to total protein concentrations.

Waddell A., Vallance J.E., Moore P.D., Hummel A.T., Wu D., Shanmukhappa S.K., Fei L., Washington M.K., Minar P., Coburn L.A., Nakae S., Wilson K.T., Denson L.A., Hogan S.P, & Rosen M.J. (2015). IL-33 signaling protects from murine oxazolone colitis by supporting intestinal epithelial function. Inflammatory bowel diseases, 21(12), 2737-2746.

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Whole Cell Lysis and Quantification

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Cells were lysed using CelLytic MT lysis buffer (Sigma Aldrich) along with the protease and phosphatase inhibitor cocktail as described elsewhere [41] . Proteins obtained were quantified using BCA reagent (Pierce). 80-100 µg for HeLa whole cell lysate and 100-120ug for Caco2 whole cell lysate from each experimental group were loaded into each well of 12.5% SDS-PAGE. Western blot transfer was performed in a semi-dry transfer apparatus Trans-Blot SD (BIORAD). Western immunoblots were developed using BCIP/ NBT (Merck) substrates against alkaline phosphatase tagged secondary antibodies.

Sarkar-Banerjee S., Chowdhury S., Sanyal D., Mitra T., Roy S.S, & Chattopadhyay K. (2018). The Role of Intestinal Fatty Acid Binding Proteins in Protecting Cells from Fatty Acid Induced Impairment of Mitochondrial Dynamics and Apoptosis. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 51(4).

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