Phosphate inhibitor cocktail 2

Manufactured by Merck Group

Sourced in United States, Denmark

The Phosphate inhibitor cocktail 2 is a laboratory reagent designed to inhibit the activity of phosphatases in biological samples. It is a concentrated solution containing a combination of phosphatase inhibitors that can be added to cell lysates, tissue homogenates, or other biological preparations to prevent the dephosphorylation of phosphorylated proteins during sample handling and analysis.

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5 protocols using phosphate inhibitor cocktail 2

Western Blot Analysis of TLR2 Expression

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Cells were washed with PBS following treatment and harvested in ice cold Tris/HCl buffer (50 mM, pH 7.4) containing EDTA (0.1 mM), EGTA (0.1 mM), and 2-mercaptoethanol (12mM), phenylmethylsulfonyl fluoride (0.2M), Sodium Orthovanadate (0.1M), Sodium fluoride (1M), protease inhibitor cocktail (1:100, Sigma Aldrich, catalog: P8340), phosphate inhibitor cocktail 2 (1:100, Sigma Aldrich, catalog: P5726), and phosphatase inhibitor cocktail 3 (1:100, Sigma Aldrich, catalog: P0044). Samples were sonicated for 3 bursts of 10 seconds, and 4X LDS sample buffer was added. Equal protein loads (20 μg) of cellular lysate were boiled and separated on a 4–12% SDS-polyacrylamide gel by electrophoresis. Rat specific anti-TLR2 (1:1000, Abcam, Cambridge, MA, USA, catalog: ab108998) and anti-β-actin antibodies (1:20,000, Sigma-Aldrich, St. Louis, MO, catalog: A3864) were used. Primary antibodies were detected using a horseradish peroxidase–conjugated antibody (TLR2: 1:10,000 anti-rabbit, Cell Signaling, Boston, MA, catalog: 7074; β-actin: 1:40,000 anti-mouse, CalBioChem, Billerica, MA, catalog:401215) and enhanced chemiluminescence (GE Healthcare, Piscataway, NJ). Relative optical densities of immunoreactivity were determined by ImageJ software (U. S. National Institutes of Health, Bethesda, Maryland, USA).

Hardigan T., Abdul Y, & Ergul A. (2016). Linagliptin reduces effects of ET-1 and TLR2-mediated cerebrovascular hyperreactivity in diabetes. Life sciences, 159, 90-96.

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Comparative Breast Cancer Cell Line Profiling

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Cell lines: Human breast cancer cell line (MCF-7), TNBC (MDA-MB-231), and murine metastatic breast cancer cell line (4T1).
Reagents: Dulbecco’s modified eagle medium (DMEM) powder, fetal bovine serum (FBS), penicillin streptomycin, trypLE Express without phenol red and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) from Gibco BRL (Carlsbad, CA, USA). DMEM media, calcium chloride dihydrate (CaCl₂·2H₂O), sodium bicarbonate (NaHCO₃), potassium phosphate monobasic (KH₂PO₄), sodium phosphate dibasic heptahydrate (H₁₅Na₂O₁₁P), bovine serum albumin (BSA), skim milk powder, tris (hydroxmethyl)amino-methane, phosphate inhibitor cocktail 2, EDTA-free protease inhibitor tablets, dimethyl sulphoxide (DMSO), and thiazolyl blue tetrazolium bromide (MTT), methanol, and trypan blue (0.4%) from Sigma-Aldrich (St. Louis, MO, USA). Sodium chloride (NaCl) and potassium chloride (KCl) from Fisher Scientific (Loughborough, Leicestershire, UK). Sodium dodecyl sulfate (SDS) and Restore PLUS Western blot stripping buffer from Thermo Fisher Scientific (Rockford, IL, USA). Triton C-100, glycine, blotting-grade blocker, Clarity Western ECL substrate, tween-20, bromophenol blue, quickstart Bradford 1X dye reagent, quickstart bovine serum albumin (BSA) standard set, dithiothreitol (DTT), and mini-protean TGX gels from Bio Rad (Hercules, CA, USA). Validated siRNAs from Qiagen (Valencia, CA, USA).

Ashaie M.A., Islam R.A., Kamaruzman N.I., Ibnat N., Tha K.K, & Chowdhury E.H. (2019). Targeting Cell Adhesion Molecules via Carbonate Apatite-Mediated Delivery of Specific siRNAs to Breast Cancer Cells In Vitro and In Vivo. Pharmaceutics, 11(7), 309.

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Transfection and Protein Extraction Protocol

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Two million cells seeded 16–18 h prior to transfection in T75 flasks. 6 μg of appropriate expression construct or appropriate empty vector (control) were transfected with 24 μl Lipofectamine reagent (Life Technologies) and 12 μl Plus reagent (Life Technologies) (17 (link)). For coexpression experiments (Supplementary Figures S4 and S7), 4 μg of appropriate expression constructs were transfected separately and together, with empty vector used as filler to bring total DNA transfected to 8 μg. Approximately 24 h post-transfection, cells were washed 1× with phosphate buffered saline (PBS) and then harvested in 500 μl Total lysis buffer (TLB: 50 mM Tris, 150 mM NaCl, 10 mM EDTA, 0.5% SDS, 0.5% Triton-X, pH 7.2) supplemented with 10 μl/ml of Halt protease inhibitor cocktail, phosphate inhibitor cocktail 2, and phosphate inhibitor cocktail 3 (Sigma). After one round of freeze (−80°C) /thaw on ice, cells were sonicated with a Microson XL-2000 sonicator (Misonix) 3× (10 s sonication/10 s rest on ice), and cell lysates were then centrifuged at 4°C at 14 000 rpm for 15 min. Protein concentrations of cleared cell lysates were determined using BioRad protein assay (Bradford method).

Christian C.M., deHaro D., Kines K.J., Sokolowski M, & Belancio V.P. (2016). Identification of L1 ORF2p sequence important to retrotransposition using Bipartile Alu retrotransposition (BAR). Nucleic Acids Research, 44(10), 4818-4834.

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Cytokine Profiling in Aortic Aneurysms

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Aneurysm tissues sampled 14 days after induction by the PPE model were lysed in Complete Mesoscale Lysis Buffer [10 mL 1x TRIS lysis buffer containing; 100 μL phosphate inhibitor cocktail #2 (Sigma-Aldrich, Søborg, Denmark), 100 μL phosphate inhibitor cocktail #3 (Sigma-Aldrich, Søborg, Denmark), and 1 tablet complete Mini, EDTA-free (Roche, Hvidovre, Denmark)] and homogenized by beads in a Tissue Lyzer II (Qiagen, Aarhus, Denmark) (Qiagen, Germany) for 3 min with maximum speed of 300 rpm and centrifuged for 20 min at +4°C at 12.000 × g. The supernatants were collected and stored at –80°C. Protein concentrations were determined using the Pierce BSA Protein Assay kit (Thermo Fischer, Roskilde, Denmark) (Bio-Rad, Copenhagen, Denmark). Cytokines and chemokines were then measured in aneurysmal protein samples and plasma samples using two different Mesoscale Discovery™ (MSD, Rockville, MA, United States) mouse pro-inflammatory V-Plex plus kits while TNFR1 and TNFR2 levels were determined using Ultra-Sensitive Kit (MSD, Rockville, MA, United States). Protocols were performed according to the manufacturer’s instructions. All samples were run in duplicate. Data were analyzed using MSD Workbench software. The cut-off threshold for inclusion of results was set at CV < 20%.

Griepke S., Grupe E., Lindholt J.S., Fuglsang E.H., Steffensen L.B., Beck H.C., Larsen M.D., Bang-Møller S.K., Overgaard M., Rasmussen L.M., Lambertsen K.L, & Stubbe J. (2022). Selective inhibition of soluble tumor necrosis factor signaling reduces abdominal aortic aneurysm progression. Frontiers in Cardiovascular Medicine, 9, 942342.

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Rapid Protein Extraction from PEG-Treated Cells

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At seven days post-subculturing, three biological replicate flasks were treated with 40% PEG-6000 (Sigma-Aldrich) in media and cells of both the mock treatment (equal volume of milliQ water) and PEG treatment were collected at 0, 10, and 30 min post-treatment. The media containing-PEG were drained off using Stericup^® filter unit (Millipore, Billerica, MA, USA), and the cells were immediately snap frozen in liquid nitrogen and stored at −80 °C until use. Approximately 3 g of cells were homogenised using T25 digital Ultra TURRAX (IKA, Staufen, Germany) and proteins were precipitated in 10% trichloroacetic acid in acetone, vortexed, and incubated overnight. Precipitated proteins were pelleted, washed, and re-suspended in urea lysis buffer (7 M urea, 2 M thiourea, and phosphate inhibitor cocktail 2 (product number P5726); Sigma-Aldrich).

Alqurashi M., Chiapello M., Bianchet C., Paolocci F., Lilley K.S, & Gehring C. (2018). Early Responses to Severe Drought Stress in the Arabidopsis thaliana Cell Suspension Culture Proteome. Proteomes, 6(4), 38.

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