Phosphatase inhibitor cocktail

Name: Phosphatase inhibitor cocktail
Brand: Roche
SKU: qiPhCZIBPBHhf-iFqxmX
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Phosphatase inhibitor cocktail is a laboratory reagent used to prevent the activity of phosphatases, which are enzymes that remove phosphate groups from proteins. This product is designed to maintain the phosphorylation state of proteins during sample preparation and analysis.

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PhosSTOP™ is a phosphatase inhibitor cocktail product manufactured by Roche. It is currently available for purchase through authorized distributors. The product is commercialized by the manufacturer, Roche.

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1 293 protocols using «phosphatase inhibitor cocktail»

Western Blot Characterization of Cellular Signaling

2025

The following antibodies were used for Western blotting: pAKT (Cell Signaling, S473; ref. 4060), AKT (Cell Signaling; ref. 9272), pS6 (Cell Signaling, S240/244; ref. 5364), S6 (Cell Signaling; ref. 2217), p4E-BP1 (Thr^37/46) (Cell Signaling; ref. 2855), 4E-BP1 (Cell Signaling; ref. 9644), Phospho-eEF2 (Thr⁵⁶) (Cell Signaling, 2331S), eEF2 Antibody (Cell Signaling, 2332S), Phospho-eEF2k (Ser³⁶⁶) (Cell Signaling; ref. 3691), eEF2k (Cell Signaling; ref. 3692), phospho-AMPKα (Thr¹⁷²) (Cell Signaling; ref. 2535), anti-AMPKα (Cell Signaling; ref: 2532S), anti-Phospho-ACC (S79) (Cell Signaling; ref. 3661), anti-ACC (Cell Signaling; ref. 3676), GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Abcam; ref. 8245), GFP (Invitrogen, A11122), anti-Puromycin (Invitrogen, MABE 343), and streptavidin-horseradish peroxidase (HRP; Invitrogen, SA10001). Tissues were homogenized in lysis buffer containing 50 mM tris, 150 mM NaCl, 10 mM MgCl₂, 0.5 mM dithiothreitol, 1 mM EDTA, and 10% (w/v) glycerol supplemented with 1% (w/v) Triton, 1% (w/v) SDS, protease inhibitor (PI; 1:500 dilution of protease inhibitor cOmplete EDTA free, Roche), and phosphatase inhibitor (1:100 dilution of phosphatase inhibitor cocktail, Roche). Lysates were cleared by centrifugation and stored at −80°C until use.
Biorthogonal noncanonical amino acid tagging (BONCAT) was performed by treating 20 μg of proteins with 75 μM Acetylene-PEG₄-Biotin (Jena Bioscience), 5.6 mM CuSO_4, 70 mM sodium ascorbate, 1 mM tris(3-hydroxypropyltriazolylmethyl)amine (THPTA; Sigma-Aldrich; ref. 762342). Biotinylated proteins were then separated by electrophoresis and immunoblotted on nitrocellulose. The resulting blot was blocked in 5% nonfat dry milk and incubated overnight with HRP-conjugated streptavidin (1:10000) or anti-GFP antibody (1:2000) in a solution of 5% nonfat dry milk. Chemiluminescence was detected using an Immobilon Classico or Crescendo western HRP substrate. Images were acquired using the ImageQuant LAS system.

Dumitras A.G., Piccoli G., Tellkamp F., Keufgens L., Baraldo M., Zorzato S., Cussonneau L., Nogara L., Krüger M, & Blaauw B. (2025). Neural stimulation suppresses mTORC1-mediated protein synthesis in skeletal muscle. Science Advances, 11(14), eadt4955.

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Liver Protein Extraction and Western Blot

2025

Homogenized livers were lysed by protein extraction solution (PRO-PREP, iNtRONBiotechnology, Korea), which included a phosphatase inhibitor cocktail (Roche, Germany) and a protease inhibitor cocktail (Calbiochem, Germany). 30 ug of total proteins were separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore, Billerica, MA). After blocking overnight with 5% skim milk, the membrane was incubated with the primary antibodies (diluted 1:1000) for 1 h at room temperature. The membranes were immunoblotted with the following primary antibodies: anti-Lcn2 (Abcam, Cambridge, MA) and anti-β-actin (Santa Cruz Biotechnology, Dallas, TX). Following the washing step with Tris-buffered saline containing 0.05% Tween-20 (TBST), the PVDF membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (diluted 1:3,000) for 1 h at room temperature. Detection of Antibody binding to the blot was conducted using enhanced chemiluminescence solution (Amersham Bioscience, United Kingdom) and X-ray film (AGFA, Belgium).

Kim J.H., Yeo I.J., Son D.J., Han S.B., Yoon D.Y., Lee D.H, & Hong J.T. (2025). Chitinase 3-like protein 1 deficiency ameliorates drug-induced acute liver injury by inhibition of neutrophil recruitment through lipocalin-2. Frontiers in Pharmacology, 16, 1548832.

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Protein Extraction and Western Blot Analysis

2025

Cell protein extraction was performed using radioimmunoprecipitation assay lysis buffer containing protease inhibitor cocktail (Roche, Burlington, MA, USA) and phosphatase inhibitor cocktail (Roche), with a reaction time of 30-60 min on ice. The protein concentration in each sample was measured using a BCA assay kit (Thermo Fisher Scientific, Rockford, IL, USA).
Western blot analysis was used with the SDS-polyacrylamide gel electrophoresis system and afterwards transferred onto PVDF membranes of 0.45-µm pore size. Then, membranes were blocked with 5% milk or Bovine Serum Albumin (Sigma-Aldrich) in 1× TBST buffer for 30-60 min. Primary antibodies including rabbit polyclonal antibodies against NLRP3, caspase-1, GSDME, interleukin-1, ASC, GAPDH (1:1,000 dilution; ABclonal, Woburn, MA, USA), and β-actin (1:3,000 dilution; Cell Signaling, Danvers, MA, USA) were incubated with the membranes overnight at 4°C for probing. On alternate days, membranes were washed with 1× TBST buffer for 10 min, repeating the process 3 times. They were then incubated with a secondary antibody conjugated to horseradish peroxidase for 60 min at 25 °C. The membranes were washed with 1× TBST buffer for 10 min, repeating the process 3 times. All cell protein levels were analyzed by Cytiva Amersham™ ECL™ Prime Western Blotting Detection Reagent (Thermo Fisher Scientific) and a ChemiDocTMXRS+ System (Bio-Rad Laboratories, Hercules, CA, USA). The intensities of the protein bands were analyzed using Image J software.

Tseng H.F., Chao H.N., Lin C.H, & Kuo C.Y. (2025). Danshensu Attenuates Palmitic Acid-Induced Activation of Hepatic Stellate Cells by Regulating Pyroptosis. International Journal of Medical Sciences, 22(8), 1865-1874.

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Quantifying Mitochondrial Protein Levels in Cell Lysates

2025

HeLa cells and skin fibroblasts were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH=8, 1 mM EGTA, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS), supplemented with Complete EDTA-free protease inhibitor cocktail (Roche Applied Science), and Phosphatase inhibitor cocktail (Roche Applied Science). Following incubation on ice for 30 min, crude extracts were centrifuged at 15000 × g for 10 min at 4 °C to remove debris, aggregates and insoluble proteins. The resulting supernatants, containing the extracted proteins were quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific), and are referred as whole cell lysates. For western blotting, 20 μg of whole cell protein extracts were dissolved in LDS sample buffer (Life Technologies) supplemented with 100 mM dithiothreitol (DTT), and heated for 5 min at 95 °C. Proteins were then separated via SDS-page electrophoresis on 4–12% Bis-Tris NuPage gels (Thermo Fisher Scientific) and transferred to a nitrocellulose membrane (Thermo Fisher Scientific) using semi-dry electrophoretic transfer (Bio-Rad). Membranes were subsequently blocked for 1 h at RT with either 5% non-fat dry milk in TBS-tween (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween) or 5% Bovine Serum Albumin (BSA) (Sigma-Aldrich) in TBS-tween, based on the primary antibody used. The membranes were then incubated with the primary antibody overnight at 4 °C. Secondary HRP-conjugated antibodies, obtained from Bio-Rad, were incubated for 1 h at RT followed by detection by chemiluminescence (SuperSignal Pico, Pierce). The following primary antibodies were used: anti-MCU (1:1000, Sigma-Aldrich HPA016480), anti-MICU1 (1:1000, Sigma-Aldrich HPA037480), anti-MICU2 (1:1000, Sigma-Aldrich HPA045511), anti-EMRE (1:1000, Santa Cruz sc-86337), anti-CLPB (1:1000, ProteinTech 15743-1-AP), anti-GRP75 (1:1000, Santa Cruz sc-133137), anti-LETM1 (1:1000, Abnova M03), anti-MITOK (1:10000, Sigma-Aldrich Cat #HPA010980), anti-OPA1 (1:1000, BD biosciences Cat #612606), anti-OXPHOS (1:1000, Abcam Cat #ab110413), anti-GAPDH (1:1000, Santa Cruz, Cat #sc-25778), anti-PDH (1:1000, Cell Signaling Technology Cat #2784), anti-AFG3L2 (1:1000, Thermo Fisher Scientific Cat #PA5-95347), anti-YME1L (1:1000, Sigma-Aldrich Cat #SAB1301252), anti-eIF2-α (1:1000, Cell Signaling Technology, Cat #5324), anti-phospho-eIF2-α (Ser51) (1:1000, Cell Signaling Technology, Cat #3597), anti-TMEM65 (1:1000, Thermo Fisher Scientific, Cat #PA5-112762, anti-Tom20 (1:1000, Cell Signaling Technology, Cat #42406).
HAP1 WT and HAP1 CLPB KO cells were washed two times with PBS and then harvested with 0.05% trypsin-EDTA (Gibco, #25300054). Immediately, the cells were lysed with RIPA buffer supplemented with protease inhibitors (leupeptin, antipain, pepstatin, 1 μg/ml of each) and PMSF 1 mM. Lysates were centrifuged at 12,000 rpm for 10 min at 4 °C to remove debris, aggregates, and insoluble proteins. The resulting supernatants, containing the extracted proteins and indicated as whole cell lysates were quantified using the DC protein assay kit (Bio-Rad, #5000113), and 25 μg of protein was loaded in a NuPAGE 10% and 4–12% Bis-Tris gels (Invitrogen, #NP0322BOX) and then separated electrophoretically in reducing and non-reducing conditions. After electrophoretic separation, proteins were transferred onto LF PVDF membranes (Bio-Rad, #1704275) and those were blocked for 1 h at room temperature, following the incubation overnight at 4 °C with the primary antibodies. The following antibodies were used: anti-CLPB (Proteintech, #15743-1-AP, 1:1000), anti-MCU (Sigma, #HPA016480, 1:500), anti-MICU1 (Sigma, #HPA037480, 1:500), anti-MICU2 (Abcam, #ab101465, 1:1000), anti-EMRE (Bethyl, #A300-BL19208, 1:1000), anti-TOM20 (Proteintech, #667771, 1: 1000), anti-HSP70 (Thermo Scientific, #MA3-028, 1:1000), and anti-OPA1 (BD Lab, #612606, 1:1000). After antibodies incubation, membranes were washed three times with TBS-Tween for 10 min and then incubated with fluorescence secondary antibodies (Licor, 1:5000) for 1 h in shaking at room temperature in the dark. Proteins were detected with chemiluminescence using the Azura Biosystems software. Proteins of interest as well as loading control bands were quantified by densitometry using ImageJ software (NIH, Bethesda, USA). Western blots shown throughout the paper are representative of at least three independent experiments.

D’Angelo D., Sánchez-Vázquez V.H., Cartes-Saavedra B., Vecellio Reane D., Cupo R.R., Delgado de la Herran H., Ghirardo G., Shorter J., Wevers R.A., Wortmann S.B., Perocchi F., Rizzuto R., Raffaello A, & Hajnóczky G. (2025). Dependence of mitochondrial calcium signalling and dynamics on the disaggregase, CLPB. Nature Communications, 16, 2810.

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Western Blot Analysis of ABCG2 Protein

2025

After treatment of QB1561 at indicated concentrations and time, cells were washed with ice-cold PBS and lysed with a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Roche, Indianapolis, Indiana, United States) for 15 min on ice. Cell debris was removed by centrifugation at 12,000 g for 15 min at 4°C. The protein lysates were analyzed by standard SDS-PAGE and transferred to a nitrocellulose membrane. The primary antibody (1:800) and the secondary antibody labeled with Horseradish Peroxidase (HRP, 1:1,500, Cell Signaling Technology, Dancers, MA, United States) were used to determine the presence of protein, ABCG2 (BXP-21, Santa Cruz Biotechnology, TX, United States) and the internal control GAPDH (Thermo Fisher Scientific, Rockford, IL, United States).

Gao H.L., Ding W., Shen Z.X, & Cui Q. (2025). Gold complex QB1561 suppresses drug-resistant cancer cells by inhibiting TrxR and mitochondrial respiratory function. Frontiers in Pharmacology, 16, 1560880.

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Top 5 most cited protocols using «phosphatase inhibitor cocktail»

Affinity Purification of SMG-9 Protein Complexes

Cited 14 times

pEF_Flag-HA-SBP-SMG-9 (2–520) (for Figure 5B), pEF_Flag-HA-SBP-NT-SMG-9 (2–181), pEF_Flag-HA-SBP-CT1-SMG-9 (185–520), pEF_Flag-HA-SBP-CT2-SMG-9 (175–520), pcDNA5/NTAP(CBP-SBP)-SMG-9 (2–520) (for Figure 5C), pSR_Strep-HA-SMG-9 full (2–520), pSR-V5-SMG-9 full (2–520), pSR_V5-NT-SMG-9 (2–181) and pSR-V5-CT-SMG-9 (182–520) were constructed by cloning each cDNA fragment by standard methods. siLentGene-puro-siSMG-9UTR (siRNA sequence targeted to the 3′-UTR of the SMG-9 mRNA: GGAGAGGAATGTCATGCAC) was constructed by method described in manual.
A 293T cells were transfected using HEKfectin (Biorad), and lysed with a loose-fit Potter–Elvehjem homogenizer in T-buffer [20 mM HEPES–NaOH at pH 7.5, 50 mM NaCl, 0.05% Tween-20, 2.5 mM MgCl₂, 0.5 mM DTT, protease inhibitor cocktail (Roche), phosphatase inhibitor cocktail (Roche) and 100 μg/ml RNaseA (Qiagen)]. The soluble fractions were pre-cleared with sepharose 4B (Sigma) and then incubated with streptavidin–sepharose (GE Biotech) for 2 h at 4°C with gentle rotation. Pre-cleared lysates were incubated with streptavidin–sepharose or anti-V5 antibodies for 2 h or 1 h at 4°C with gentle rotation. For antibodies, subsequently, the soluble fractions were incubated with 30 μl of protein G sepharose (GE Biotech) for an additional 1 h at 4°C with gentle rotation. After washing with RNase(−) T-lysis buffer, the affinity-purified protein complexes were eluted by incubation at 4°C for 30 min with RNase(−) lysis buffer containing 2 mM biotin (Sigma) or SDS sample buffer, respectively. All proteins in western blot experiments were detected with an ECL western blot detection kit (GE Biotech) or Lumi-Light (Roche). All experiments were performed two to three times, and typical results are shown.

Fernández I.S., Yamashita A., Arias-Palomo E., Bamba Y., Bartolomé R.A., Canales M.A., Teixidó J., Ohno S, & Llorca O. (2010). Characterization of SMG-9, an essential component of the nonsense-mediated mRNA decay SMG1C complex. Nucleic Acids Research, 39(1), 347-358.

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Corresponding organizations : Consejo Superior de Investigaciones Científicas, Unidades Centrales Científico-Técnicas

SILAC-Based Proteomic Analysis of HeLa Cells

Cited 11 times

Gibco SILAC DMEM basal cell culture medium (Invitrogen, Carlsbad, CA) containing 2 mM L-Glutamine, 10% dialyzed fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) and 100 U/ml penicillin and streptomycin was supplemented with 100 mg/L L-Lysine and 20 mg/L L-Arginine or 100 mg/L [U-¹³C₆]-L-Lysine and 20 mg/L [U-¹³C₆, ¹⁵N₄]-L-Arginine (Invitrogen, Carlsbad, CA) to make the “Light” SILAC or “Heavy” SILAC culture media, respectively. HeLa cells were obtained from ATCC and were propagated in SILAC medium for more than nine generations to ensure nearly 100% incorporation of labeled amino acids before the experiment was performed. After being washed with precooled PBS buffer, HeLa cells were lysed in E1A (250 mM NaCl, 50 mM HEPES (pH 7.5), 0.1% NP40, 5 mM EDTA, protease inhibitor cocktail (Roche, Indianapolis, IN) and phosphatase inhibitor cocktail (Roche, Indianapolis, IN)) or RIPA (1X PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml PMSF, 1mM sodium orthovanadate, protease inhibitor cocktail (Roche, Indianapolis, IN) and phosphatase inhibitor cocktail (Roche, Indianapolis, IN)) lysis buffer respectively. “Light” cell lysate and “Heavy” lysate were mixed at a 1:1 ratio, and the mixed lysates were incubated with the precipitating antibody for 8 hr, followed by 16 hr incubation with protein G beads (Invitrogen, Carlsbad, CA). Immune complexes were washed three times in lysis buffer (E1A or RIPA respectively) and once in sterile water, and then incubated in 8 M urea for 30 min at room temperature. The supernatant was reduced with DTT, and then alkylated with iodoacetamide. The resulting samples from E1A or RIPA lysis buffer were dialyzed against 2 M urea/100 mM NH₄HCO₃ at 37°C for 5 hr and then analyzed by Multidimensional Liquid Chromatography Coupled with Tandem Mass Spectrometry (MudPIT) twice. Since the existence of the stable (heavy) isotope in nature is less than 1%, any “light” peptide detected by MudPIT with no similar amount of “heavy” peptide detected was considered as a contaminant.

Yang Q., Deng X., Lu B., Cameron M., Fearns C., Patricelli M.P., Yates JR I.I.I., Gray N.S, & Lee J.D. (2010). Pharmacological Inhibition of BMK1 Suppresses Tumor Growth Through PML. Cancer cell, 18(3), 258-267.

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Corresponding organizations : Scripps Research Institute, Dana-Farber Cancer Institute, Harvard University, Torrey Pines Institute For Molecular Studies

Proteomic Analysis of Xenograft Tumor Tissue

Cited 7 times

Xenograft-derived tissue was harvested and frozen at −80 °C prior to use. Small pieces of tumor tissue were added to 2 ml tubes with ceramic beads together with ice-cold lysis buffer containing 1% Triton X-100, 50 mM Hepes pH 7.4, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 100 mM NaF, 10 mM NaPPi, 10% glycerol, 1 mM Na₃VO₄, Complete Protease Inhibitor Cocktail and Phosphatase Inhibitor cocktail (Roche Diagnostics). Protein supernatants were isolated as described previously [27 (link)], and protein concentration was determined by BCA assay (Pierce). Samples were diluted to a uniform protein concentration, and then denatured in 1% SDS sample buffer for 5 minutes at 95°C. Samples were stored at −80°C until use. RPPA analysis was performed as described previously [27 (link), 28 (link)]. Data were obtained for 161 antibodies. A logarithmic value reflecting the relative amount of each protein in each sample was generated for analyses [29 (link)]. Similarity of proteomic gene expression was evaluated using cluster analysis as well as by Pearson distance correlation analysis. An RPPA data summary is shown in Supplemental Table 6. Sample information for the RPPA data is shown in Supplemental Table 7, designated “rppa.info.final.color”. Supplemental Tables 6 and 7 are available by download only from www.bcxenograft.org.

Zhang X., Claerhout S., Pratt A., Dobrolecki L.E., Petrovic I., Lai Q., Landis M.D., Wiechmann L., Schiff R., Giuliano M., Wong H., Fuqua S.W., Contreras A., Gutierrez C., Huang J., Mao S., Pavlick A.C., Froehlich A.M., Wu M.F., Tsimelzon A., Hilsenbeck S.G., Chen E.S., Zuloaga P., Shaw C.A., Rimawi M.F., Perou C.M., Mills G.B., Chang J.C, & Lewis M.T. (2013). A Renewable Tissue Resource of Phenotypically Stable, Biologically and Ethnically Diverse, Patient-derived Human Breast Cancer Xenograft (PDX) Models. Cancer research, 73(15), 4885-4897.

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Corresponding organizations : Baylor College of Medicine, The University of Texas MD Anderson Cancer Center

Purification of Dynein, Nudel, and Lis1 Proteins

Cited 7 times

Cultures of S. cerevisiae for protein purification were grown, harvested, and frozen as previously described (Reck-Peterson et al., 2006 (link)). Dynein constructs were purified via their ZZ tag, labeled with HaloTag-TMR or HaloTag-PEG-biotin ligands (Promega), and eluted into a modified TEV buffer (50 mM Tris-HCl [pH 8.0], 150 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 10% glycerol, 1 mM DTT, 1 mM PMSF, and 0.1 mM Mg-ATP). Nudel was similarly purified via its ZZ tag, with the exception that the lysis buffer additionally contained a phosphatase inhibitor cocktail (Roche), and all buffers lacked Mg-ATP.
Lis1 constructs were purified via His₈ and ZZ tags. Lysed cells were resuspended in Buffer A (final concentrations: 50 mM potassium phosphate [pH 8.0], 150 mM potassium acetate, 150 mM NaCl, 2 mM magnesium acetate, 5 mM β-mercaptoethanol, 10% glycerol, 0.2% Triton X-100, 0.5 mM Pefabloc, and 1 mM PMSF) supplemented with 10 mM imidazole (pH 7.5). Subsequent steps were at 4°C unless indicated. The lysate was clarified by centrifugation at 264,900 g for 1 hr. The supernatant was incubated with Ni-NTA agarose (QIAGEN) for 1 hr, transferred into a column, washed three times with Buffer A + 20 mM imidazole, and eluted with Buffer A + 250 mM imidazole. Eluted protein was then incubated with IgG sepharose beads (Amersham Pharmacia) for 1 hr, transferred into a column, and washed twice with Buffer A + 20 mM imidazole and once with TEV buffer (10 mM Tris-HCl [pH 8.0], 150 mM KCl, 10% glycerol, 0.2% Triton X-100, 1 mM PMSF, and 1 mM DTT). Lis1 was released from beads via incubation with TEV protease for 1 hr at 16°C, resulting in cleavage from the His₈-ZZ tag.

Huang J., Roberts A.J., Leschziner A.E, & Reck-Peterson S.L. (2012). Lis1 Acts as a “Clutch” between the ATPase and Microtubule-Binding Domains of the Dynein Motor. Cell, 150(5), 975-986.

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Corresponding organizations : Harvard University, University of Leeds

Phosphoproteomics of Hyper-Virulent M. tuberculosis

Cited 6 times

A previously described hyper-virulent clinical Beijing genotype Mycobacterium tuberculosis isolate, SAW5527, isolated from a TB patient attending a primary health care clinic in the Western Cape province, South Africa was used for this phosphoproteomics analysis (de Souza et al., 2010 (link)). Secondary cultures were inoculated into 50 ml 7H9 Middlebrooks medium supplemented with Dextrose and Catalase and incubated at 37°C until early-logarithmic phase (OD₆₀₀ between 0.6 and 0.7). Mycobacterial cells were collected by centrifugation (2000 × g for 10 min at 4°C) and washed two times with cold lysis buffer containing 10 mM Tris-HCl (pH 7.4), 0.1% Tween-80, Complete Protease inhibitor cocktail (Roche, Mannheim Germany) and Phosphatase inhibitor cocktail (Roche, Mannheim Germany). An equal amount of 0.1 mm glass beads (Biospec Products Inc., Bartlesville, OK) was added to the cell pellet after centrifugation together with cold 300 μl lysis buffer and 10 μl DNaseI (2U/ml) (NEB, New England Laboratories). Lysis was achieved by mechanical bead-beating in a Rybolyser (Bio101 SAVANT, Vista, CA) for 6 cycles of 20 s at a speed of 4.0 m.s⁻¹, with 1 min cooling periods on ice. The whole cell lysates were filter-sterilized with a sterile 0.22 μm pore acrodisc 25 mm PF syringe filter (Pall Life Sciences, Pall Corporation, Ann Arbour, MI) and stored at −80°C. The protein concentration of the whole cell lysate was determined using the RC DC Protein assay according to manufacturer's instructions (BioRad). A single biological replicate was analyzed in triplicate for downstream phosphoproteomic analysis.

Fortuin S., Tomazella G.G., Nagaraj N., Sampson S.L., Gey van Pittius N.C., Soares N.C., Wiker H.G., de Souza G.A, & Warren R.M. (2015). Phosphoproteomics analysis of a clinical Mycobacterium tuberculosis Beijing isolate: expanding the mycobacterial phosphoproteome catalog. Frontiers in Microbiology, 6, 6.

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Corresponding organizations : South African Tuberculosis Vaccine Initiative, Stellenbosch University, South African Medical Research Council, University of Bergen, Max Planck Society, University of Cape Town, University of Oslo

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Phosphatase inhibitors - 1485 citations Cocktail inhibitor - 10 citations Cocktails of protease and phosphatase inhibitors - 7 citations Protease and phosphatases inhibitor cocktails - 6 citations Proteases and phosphatases inhibitor cocktails - 4 citations Inhibitor cocktails - 3 citations Phosphatases inhibitors - 3 citations Phosphatases - 3 citations Phosphatases inhibitors cocktail - 3 citations Cocktails of protease inhibitors and phosphatase inhibitors - 2 citations

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