Cellytic m

Manufactured by Merck Group

Sourced in United States, United Kingdom, Germany

CelLytic M is a cell lysis reagent that enables the extraction of proteins from mammalian cells. It is designed to disrupt cell membranes and solubilize cellular proteins, making them accessible for further analysis or purification.

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CelLytic M mammalian cell lysis/extraction reagent (16 citations) CelLytic M Cell Lysis Reagent (136 citations) CelLytic M lysis buffer (40 citations) CelLytic™ M mammalian cell lysis reagent (2 citations) CelLytic-M kit (2 citations) CelLytic M lysis reagent (15 citations) CelLytic™ M Cell Lysis (2 citations) CelLytic M solution (2 citations) CelLytic M Cell Lysis/Extraction Reagent (2 citations) CelLytic M Cell Lysis buffer (13 citations)

165 protocols using cellytic m

Mitochondrial Protein Analysis in Tissues

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Flash frozen powdered quadriceps and heart tissue, as well as isolated mitochondrial pellets from each tissue were
thawed on ice and homogenized in CelLytic M (Sigma-Aldrich; Cat# C2978) supplemented with protease inhibitor cocktail and 10
mM nicotinamide using a motor-drive Potter-Elvehjem tissue grinder. Samples were centrifuged at 14,000 × g for 10 min
at 4°C and the supernatant saved and frozen at −80°C until later analysis. Protein concentration was
determined via the BCA method and the samples were diluted in CelLytic M buffer. Forty micrograms of protein sample were
combined with 5× loading buffer and resolved by SDS-PAGE, transferred to nitrocellulose, blocked for ~1 hr in 5%
milk prepared with TBS followed by western blotting with specific antibodies. Antibodies employed herein were: MCD
(Proteintech; #15265-1-AP), Sirt5 (see Key Resources Table), malonyl-lysine (Cell
Signaling; #14942), ETFDH (Abcam; #ab126576), ETFα (Abcam; #ab110316), Hadha (Abcam; #ab203114), OXPHOS cocktail
(Abcam; #ab110413), Pdhe1α (Abcam; #ab168379), phosphorylated Pdhe1α (Serine 232; #AP1063, Serine 293;
#AP1062).

Fisher-Wellman K.H., Draper J.A., Davidson M.T., Williams A.S., Narowski T.M., Slentz D.H., Ilkayeva O.R., Stevens R.D., Wagner G.R., Najjar R., Hirschey M.D., Thompson J.W., Olson D.P., Kelly D.P., Koves T.R., Grimsrud P.A, & Muoio D.M. (2019). Respiratory Phenomics across Multiple Models of Protein Hyperacylation in Cardiac Mitochondria Reveals a Marginal Impact on Bioenergetics. Cell reports, 26(6), 1557-1572.e8.

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Western Blot Analysis of Cellular Signaling

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Following cellular treatment, protein extraction, quantification, and Western blot analysis were performed as previously described [26 (link), 28 , 29 (link)]. Briefly, cells were lysed using the mammalian cell lysis buffer CelLytic M (Sigma, St Louis, MO, USA), and protein quantified using the bicinchoninic acid assay as we previously described [26 (link), 28 , 29 (link)]. Protein (35 µg) was fractionated in a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Membranes were blocked for 1 hour with SuperBlock T-20 (Pierce, Rockford, IL, USA) and blotted for 2 hours or overnight with monoclonal antibodies to TLR3, claudin-5 (Abcam), JNK, phopho-JNK, c-Jun, phospho-c-Jun (all from Cell Signaling Technology, Danvers, MA, USA). These primary antibodies were used at 1:1,000 dilutions. Membranes were then washed, blotted for 1 hour with horseradish peroxidase-conjugated secondary antibody, washed again, and visualized using the enhanced chemiluminescence (Pierce) and gel doc system (Syngene, Frederick, MD, USA). After each western blot experiment, membranes were stripped using the Restore Western Blot Stripping Buffer (Pierce) and re-blotted with β-actin antibody (Abcam) to confirm equal loading.

Bhargavan B, & Kanmogne G.D. (2017). Toll-Like Receptor-3 Mediates HIV-1-Induced Interleukin-6 Expression In The Human Brain Endothelium Via TAK1 And JNK Pathways: Implications For Viral Neuropathogenesis. Molecular neurobiology, 55(7), 5976-5992.

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Arrestin-Rhodopsin Cross-Linking Analysis

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The open reading frames of full-length arrestin with C-terminal FLAG tag and full length rhodopsin with C-terminal HA tag were cloned into pcDNA6. Cysteine mutations (41 for arrestin and 51 for rhodopsin) were systematically introduced into arrestin and rhodopsin in these two DNA vectors. AD293 cells were split one day before transfection at 50,000 cells/well in a 24-well plate. Cells were grown for one day, then transfected with 100 ng rhodopsin constructs (pcDNA6-rho-3HA) plus 100 ng arrestin plasmid (pcDNA6-Arr-3FLAG) by Lipofectamine 2000 (DNA/Lipofectamine 2000 ratio of 1:2) in each well. Cells were grown for 2 days after transfection, and were then treated at room temperature with H₂O₂, which was freshly diluted in the cell culture medium to a final concentration of 1 mM. After 5 min treatment with H₂O_2, the medium was aspirated and 100 μl of CelLytic M (Sigma C2978) were added to each well and the plate was shaken for 10 min at room temperature. Cell lysates were transferred to 1.5 ml tubes and spun at 16,000 x g at 4 °C for 5 min. The supernatants (10 μl) were mixed with an equal volume of 2×SDS loading buffer (without reducing agents) for 5 min at room temperature, and loaded onto a protein gel for Western blot analysis. HRP-conjugated anti-FLAG (Sigma M2) and anti-HA (Sigma) antibodies were used to probe for free and cross-linked arrestin and rhodopsin proteins.

Kang Y., Zhou X.E., Gao X., He Y., Liu W., Ishchenko A., Barty A., White T.A., Yefanov O., Han G.W., Xu Q., de Waal P.W., Ke J., Eileen Tan M.H., Zhang C., Moeller A., West G.M., Pascal B., Van Eps N., Caro L.N., Vishnivetskiy S.A., Lee R.J., Suino-Powell K.M., Gu X., Pal K., Ma J., Zhi X., Boutet S., Williams G.J., Messerschmidt M., Gati C., Zatsepin N.A., Wang D., James D., Basu S., Roy-Chowdhury S., Conrad C., Coe J., Liu H., Lisova S., Kupitz C., Grotjohann I., Fromme R., Jiang Y., Tan M., Yang H., Li J., Wang M., Zheng Z., Li D., Howe N., Zhao Y., Standfuss J., Diederichs K., Dong Y., Potter C.S., Carragher B., Caffrey M., Jiang H., Chapman H.N., Spence J.C., Fromme P., Weierstall U., Ernst O.P., Katritch V., Gurevich V.V., Griffin P.R., Hubbell W.L., Stevens R.C., Cherezov V., Melcher K, & Xu H.E. (2015). Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature, 523(7562), 561-567.

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Measuring Cellular Oxidation and Proliferation

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Reactive oxygen species were measured using CellROX Green Reagent (Invitrogen). All samples were imaged under the same parameters, and signal intensity in each field was measured using ImageJ software. For proliferation, 0.2 μM BrdU was added in the medium for 6 or 12 hours, after which the cultures were fixed and incubated with 2 N HCl for 20 min before immunostaining. For Western blotting, cells or mouse cortical tissue was lysed in CelLytic M (Sigma-Aldrich) and protease inhibitors (Roche), boiled in SDS–polyacrylamide gel electrophoresis loading buffer plus β-mercaptoethanol for 5 min, and loaded at 30 μg per lane for human cells or 10 μg for brain lysate. Membranes were incubated with the following antibodies; p44/42 MAPK (pERK) (Cell Signaling Technology, 9102), GAPDH (Sigma-Aldrich, G8795), GFAP (Chemicon, MAB360), neurocan (R&D Systems, AF5800), and brevican (BD Biosciences, 610894). The H-Ras Activation Assay Kit (Cell Biolabs Inc.), Periostin Elisa Kit (Boster, EK0985), and Senescence β-Galactosidase Staining Kit (Cell Signaling Technologies, 9860) were used according to the manufacturers’ instructions.

Krencik R., Hokanson K.C., Narayan A.R., Dvornik J., Rooney G.E., Rauen K.A., Weiss L.A., Rowitch D.H, & Ullian E.M. (2015). Dysregulation of astrocyte extracellular signaling in Costello syndrome. Science translational medicine, 7(286), 286ra66.

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Purification of Viral Proteins

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HEK293 were transfected with the appropriate expression vectors using liposomal reagent Transfast^TM following manufacturer’s instructions, washed and lysed 48 h later with CelLytic M (Sigma-Aldrich, USA). Lysates were cleared by centrifugation.
For purification of HA-tagged pUL56, the cell-free reaction was performed with Anti-HA Immunoprecipitation Kit according to the manufacturer’s protocol (Sigma-Aldrich, USA).
For purification of His-tagged pUL89, the cell-free reaction was performed with Ni resin (Clontech, USA).
All proteins were concentrated approximately 5-fold using Pall centrifugal filters (Pall, USA), and protein concentration was determined by the Bradford method using bovine serum albumin (Sigma-Aldrich, USA) as standard protein.

Ligat G., Jacquet C., Chou S., Couvreux A., Alain S, & Hantz S. (2017). Identification of a short sequence in the HCMV terminase pUL56 essential for interaction with pUL89 subunit. Scientific Reports, 7, 8796.

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Quantification of Tumor Chemokine Profiles

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Tumors collected at indicated treatment time points were minced, lysed in CelLytic M (Sigma, Cat#C2978) containing halt protease and phosphatase inhibitor (Thermo Fisher, Cat#78446) in a 1:100 ratio, and incubated on ice for 30min with intermittent vortexing. Tumor lysates were assayed for raw protein concentration with Coomassie assay (Pierce, Cat#23200). IL-8 (BD Bioscience, Cat#555244), CXCL-2, and Cxcl15 (R&D Systems, Cat#DY27605 and Cat#DY442) were analyzed by ELISA kits following the manufacturer’s instructions. IL-8, CXCL-1, CXCL-5, and CXCL-12 (MSD, Cat#K15067L-1) were evaluated using the mesoscale MESO QuickPlex SQ 120 instrument.

Lopez-Bujanda Z.A., Haffner M.C., Chaimowitz M.G., Chowdhury N., Venturini N.J., Patel R.A., Obradovic A., Hansen C.S., Jacków J., Maynard J.P., Sfanos K.S., Abate-Shen C., Bieberich C.J., Hurley P.J., Selby M.J., Korman A.J., Christiano A.M., De Marzo A.M, & Drake C.G. (2021). Castration-mediated IL-8 Promotes Myeloid Infiltration and Prostate Cancer Progression. Nature cancer, 2(8), 803-818.

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Time Course of ATF3 Protein Expression

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The time course of the expression of ATF3 protein was determined by
the capillary electrophoresis immunoassay using the Simple Western system as
described previously. In brief, L4/L5 naïve or injured DRGs were
collected 1, 3, and 7 days post-SNL. Tissues were homogenized in CelLytic M
(Sigma; C2978) containing a protease inhibitor cocktail (Sigma; 4693159001).
The lysate protein concentration was determined using the Pierce BCA protein
assay kit (Thermo Scientific; 23227). Equal amounts of DRG lysates (1
μg) were mixed with Simple Western reagents and loaded to each
capillary. The primary antibodies were diluted with antibody diluent
(ProteinSimple) at 1:50 for ant-ATF3 (Santa Cruz; sc-188), 1:50 for
anti-Mettl14 (Proteintech; 26158-1-AP), and 1:50 for anti-GAPDH (Abcam;
ab9485).

Weng Y.L., Wang X., An R., Cassin J., Vissers C., Liu Y., Liu Y., Xu T., Wang X., Wong S.Z., Joseph J., Dore L.C., Dong Q., Zheng W., Jin P., Wu H., Shen B., Zhuang X., He C., Liu K., Song H, & Ming G.L. (2018). Epitranscriptomic m6A Regulation of Axon Regeneration in the Adult Mammalian Nervous System. Neuron, 97(2), 313-325.e6.

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Western Blotting and Immunoprecipitation Workflow

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Cells were washed twice with cold PBS and suspended with CelLyticM (Sigma-Aldrich) containing protease and phosphatase inhibitors (Sigma-Aldrich). After incubation, lysates were centrifuged and the supernatants were mixed with 2×sample buffer. Proteins were separated by SDS-PAGE, and transferred to polyvinyl difluoride (PVDF) membranes (Millipore, Billerica, MA). Membranes were blocked with SuperBlock (TBS) blocking buffer (Thermo Fisher Scientific, Waltham, MA) and incubated with the indicated antibodies. After incubation with anti-mouse or anti-rabbit horseradish peroxidase antibodies, ECL Western Blotting Detection Reagents (GE Healthcare) was used to detect the signal.
For immunoprecipitation, supernatants were incubated with Myc-tag antibody (Sepharose Conjugate) or anti-FLAG M2 affinity gel. After centrifugation and washing with TBS-T, the protein-beads complexes were separated and subjected to immunoblotting as described above.

Chen F., Ishikawa Y., Akashi A., Naoe T, & Kiyoi H. (2016). Co-expression of wild-type FLT3 attenuates the inhibitory effect of FLT3 inhibitor on FLT3 mutated leukemia cells. Oncotarget, 7(30), 47018-47032.

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Mitochondrial Respiration and Glycolysis Profiling

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OCR, a measure of mitochondrial respiration, and ECAR, a measure of glycolysis, were determined using an XF96 extracellular flux analyzer (Seahorse Bioscience). Transfected 3T3-L1 cells were seeded in a 96-well XF96 cell culture microplate (Seahorse Bioscience) at a density of 7,000 cells per well in 200 μl of DMEM (4.5 g/l glucose) supplemented with 10% FBS (Sigma) 36 h before measurement. Six replicates per cell type were included in the experiments, and four wells were chosen evenly in the plate to correct for temperature variation. On the day of the assay, the growth medium was exchanged for assay medium (unbuffered DMEM with 4.5 g/l glucose). Oligomycin at a final concentration of 2.0 μM, FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) at 1.0 μM, 2-deoxyglucose at 100 mM, and rotenone at 15.0 μM were sequentially injected into each well in accordance with the manufacturer’s protocol. Basal mitochondrial respiration, maximal respiration, ATP production, and basal glycolysis were determined according to the manufacturer’s instructions. At the conclusion of the assay, cells in the analysis plate were lysed using CelLytic M (Sigma). Protein concentration was measured using the Bradford assay^{59 (link)} and used to normalize the bioenergetic profile data.

Minster R.L., Hawley N.L., Su C.T., Sun G., Kershaw E.E., Cheng H., Buhule O.D., Lin J., Reupena M.S., Viali S., Tuitele J., Naseri T., Urban Z., Deka R., Weeks D.E, & McGarvey S.T. (2016). A thrifty variant in CREBRF strongly influences body mass index in Samoans. Nature genetics, 48(9), 1049-1054.

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Quantifying Cellular Ferritin Levels

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Following 24 h incubation with the bread digestions, the cellular medium was aspirated, the cells were rinsed with 18 Ω MilliQ H₂O and subsequently lysed by scraping in 200 μl of Cellytic M (Sigma-Aldrich, UK). Cell pellets were kept on ice for 15 min and stored at −80 °C. For analysis, samples were thawed and centrifuged at 14,000g for 15 min. Cellular debris was discarded and the supernatant containing the proteins was used for ferritin determination using the Spectro Ferritin ELISA assay (RAMCO, USA). The ferritin concentration in the samples was determined using a microplate reader at an excitation wavelength of 500 nm according to the manufacturer’s protocol. Ferritin concentrations were normalised to total cell protein using the Pierce Protein BCA protein assay (ThermoFisher Scientific, UK).

Rodriguez-Ramiro I., Brearley C.A., Bruggraber S.F., Perfecto A., Shewry P, & Fairweather-Tait S. (2017). Assessment of iron bioavailability from different bread making processes using an in vitro intestinal cell model. Food Chemistry, 228, 91-98.

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