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Phostop phosphatase inhibitor cocktail

Manufactured by Roche
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Sourced in Germany, Switzerland

PhoSTOP is a phosphatase inhibitor cocktail. It is designed to inhibit phosphatase activity in cellular and biochemical assays. The product contains a proprietary blend of phosphatase inhibitors.

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Lab products found in correlation

Ripa buffer, by Boston BioProducts (3 mentions) Complete mini edta free protease inhibitor cocktail, by Roche (3 mentions) Iblot transfer stack, by Thermo Fisher Scientific (3 mentions) Complete ultra protease inhibitor, by Roche (3 mentions) Bolt 10 bis tris plus gel, by Thermo Fisher Scientific (3 mentions) Iblot2 system, by Thermo Fisher Scientific (3 mentions) Novex mini cell, by Thermo Fisher Scientific (3 mentions) Ripa buffer, by Merck Group (3 mentions) Ecl plus western blotting detection system, by GE Healthcare (3 mentions) Complete protease inhibitor cocktail, by Roche (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Immobilon p polyvinylidene difluoride membranes, by Bio-Rad (2 mentions) Enhanced chemi luminescence (ecl), by GE Healthcare (2 mentions) Py705 stat3, by Cell Signaling Technology (2 mentions) α tubulin, by Abcam (2 mentions) Bioruptor 200, by Diagenode (2 mentions) Tautomycin, by Enzo Life Sciences (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Trypsin edta, by Merck Group (1 mentions)

Close spelling variants of this product

PhoSTOP phosphatase inhibitor (13 citations) Phosphatase inhibitor cocktail (633 citations) Inhibitor cocktails (2 citations) Cocktail inhibitor (7 citations) Phosphatase inhibitors (745 citations) PhoSTOP (88 citations) Phosphatases (2 citations)

13 protocols using phostop phosphatase inhibitor cocktail

1

Cell Culture Media Composition

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Dulbecco’s modified Eagle’s medium (DMEM), glutamine, trypsin-EDTA were from Sigma-Aldrich (Castle Hill, New South Wales, Australia). Penicillin/streptomycin was from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) was from Life Technologies (Melbourne, Victoria, Australia). Protease Inhibitor Cocktail Set III was from Merck Millipore (Darmstadt, Germany) and PhoSTOP Phosphatase Inhibitor Cocktail was from Roche Diagnostics Australia (Castle Hill, NSW, Australia). Tautomycin was from Enzo Life Science (ALX-380-041-CO25, Michigan, USA).
McMahon K.A., Wu Y., Gambin Y., Sierecki E., Tillu V.A., Hall T., Martel N., Okano S., Moradi S.V., Ruelcke J.E., Ferguson C., Yap A.S., Alexandrov K., Hill M.M, & Parton R.G. (2019). Identification of intracellular cavin target proteins reveals cavin-PP1alpha interactions regulate apoptosis. Nature Communications, 10, 3279.
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2

Investigating EGFR Signaling in PTPN2-Expressing HeLa Cells

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HeLa cells expressing FKBP12F36V-PTPN2 were seeded into 10-cm culture dishes until 80% confluency. All dishes were then washed with PBS once and treated with serum-free medium together with DMSO, GePhos1 (500 nM), inactive GePhos1 (500 nM), or gefitinib (500 nM) (three replicates for each group) for 24 hours. At the last 15 min, cells were then treated with EGF (final 100 ng ml−1) for 7.5 or 15 min. No EGF treatment was applied for 0-min sample. To collect samples for mass spectrometry, all medium were removed, rinsed with cold PBS once, and immediately snap-frozen by liquid nitrogen. All cells were then scraped down by cell scrapers using 500 μl of 10 M urea containing cOmplete protease inhibitor cocktail (Roche, #11697498001) and PhoSTOP phosphatase inhibitor cocktail (Roche, #4906845001), snap-frozen in liquid nitrogen, and store at −80°C until sample processing. Three replicates were used for each condition.
Hu Z., Chen P.H., Li W., Krone M., Zheng S., Saarbach J., Velasco I.U., Hines J., Liu Y, & Crews C.M. (2024). EGFR targeting PhosTACs as a dual inhibitory approach reveals differential downstream signaling. Science Advances, 10(13), eadj7251.
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3

Preparation of Postsynaptic Density Fractions

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PFC tissues were dissected from 8 week old rats as previously described (Spijker, 2011 (link)). PSDs were prepared as follows; tissue samples from WT and Shank3 KO rats were homogenized in 2-[4-(2-hydroxyethyl)piperazin-1-yl]-ethanesulfonic acid (HEPES)-A containing 4 mM HEPES, pH 7.4, 0.32 M sucrose, Protease Inhibitor Cocktail and PhoSTOP Phosphatase Inhibitor Cocktail (both from Roche). Nuclear fractions were then precipitated by centrifuging twice at 700 g for 15 min, and the resulting supernatants were further centrifuged at 21,000 g for 15 min. The precipitates were re-suspended in HEPES-B containing 4 mM HEPES, pH 7.4, Protease Inhibitor Cocktail and PhoSTOP Phosphatase Inhibitor Cocktail, homogenized and rotated at 4°C for 1 hr. These lysates were centrifuged at 32,000 g for 20 min and washed twice with HEPES-C containing 50 mM HEPES, pH 7.4, 0.5% Triton X-100, Protease Inhibitor Cocktail and PhoSTOP Phosphatase Inhibitor Cocktail. Finally, postsynaptic density fractions were resuspended in HEPES-C containing 1.8% sodium dodecyl sulfate (SDS) and 2.5 M urea. Protein concentrations were determined using the Pierce BCA protein assay.
Harony-Nicolas H., Kay M., du Hoffmann J., Klein M.E., Bozdagi-Gunal O., Riad M., Daskalakis N.P., Sonar S., Castillo P.E., Hof P.R., Shapiro M.L., Baxter M.G., Wagner S, & Buxbaum J.D. (2017). Oxytocin improves behavioral and electrophysiological deficits in a novel Shank3-deficient rat. eLife, 6, e18904.
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4

Co-Immunoprecipitation of Protein Complexes

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For co-immunoprecipitation experiments, HeLa cells were grown in 10 cm Petri dishes and transfected with the indicated constructs as described above. After 36 hours, cells were lysate in an appropriate volume of lysis buffer (125 mM NaCl, 25 mM TRIS-Cl pH7.4, 1 mM EGTA-Tris pH 7.4, 0.5% n-Dodecyl β-D-maltoside, Complete EDTA free protease inhibitor mixture and PhoSTOP Phosphatase Inhibitor Cocktail (Roche Applied Science)). Whole cell lysate was precleared for 30’ with a control agarose resin (Pierce). 1 mg of pre-cleared proteins from the indicated conditions was incubated with monoclonal αFlag or αHA agarose-conjugated antibody (Sigma-Aldrich) and the co-immunoprecipitation was performed following manufacturer instructions. After three 10’ washes in lysis buffer, the bait was eluted in a non-reducing Laemli sample buffer and denatured for 10’ at 90°C. The precleared lysate (Input) and the eluted fraction (CoIP) were separated by SDS-PAGE, transferred to Hybond-C Extra membrane (Amersham) and developed according to standard procedures. The same membrane in each panel was stripped for 10’ in StripABlot stripping buffer (Euroclone) and probed with different antibodies as indicated.
Patron M., Granatiero V., Espino J., Rizzuto R, & De Stefani D. (2018). MICU3 is a tissue-specific enhancer of mitochondrial calcium uptake. Cell Death and Differentiation, 26(1), 179-195.
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5

Protein Extraction and Western Blotting

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Cells were lysed with RIPA buffer (Boston Bioproducts) supplemented with cOmplete Mini EDTA-free Protease inhibitor cocktail (Roche) and PhoSTOP phosphatase inhibitor cocktail (Roche). The total cell lysate (20μg) was subjected to SDS polyacrylamide gel electrophoresis and transferred to Immobilon-P polyvinylidene difluoride membranes (Bio-Rad Laboratories). All antibodies are listed in supplementary table 1.
Cooper A.J., Kobayashi Y., Kim D., Clifford S.E., Kravets S., Dahlberg S.E., Chambers E.S., Li J., Rangachari D., Nguyen T., Costa D.B., Rabin M.S., Wagle N., Sholl L.M., Jänne P.A, & Oxnard G.R. (2020). Identification of a RAS-activating TMEM87A-RASGRF1 fusion in an exceptional responder to sunitinib with non-small cell lung cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(15), 4072-4079.
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6

Protein Extraction and Western Blot Analysis

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Cells were lysed for total protein extraction using a radio immunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl buffer, 1% Triton X-100, 150 mM NaCl, 0.1% SDS, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% sodium deoxycholate) in the presence of Protease Inhibitor Cocktail and PhoSTOP Phosphatase Inhibitor Cocktail (both from Roche, Basel, Switzerland) for 30 min, then cleared by centrifugation at 4°C. Nuclear extracts were isolated with a Nuclear and Cytoplasmic Protein Extraction Kit (Bioteke, Beijing, China), according to the manufacturer's instructions. Both total and nuclear extracts were quantified using a BCA assay kit (KeyGEN, Nanjing, China), then resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. These membranes were the blotted with appropriate primary antibodies and horseradish peroxidase-coupled secondary antibodies, and visualized using chemiluminescence (DNR Bio-Imaging Systems, Jerusalem, Israel) and x-ray films. For Western Blot analysis, β-tubulin and lamin B were used as loading control. Antibodies were obtained from various sources, including Sigma–Aldrich (anti-β-tubulin, anti-β-catenin and anti-CCNG2), Abcam (Cambridge, MA, USA) (anti-CCNG2 and anti-Runx2), Sangon Biotech (anti-ALP and anti-lamin B) and Santa Cruz (anti-CCND1 and anti-c-Myc).
Gao J., Liu Q., Liu X., Ji C., Qu S., Wang S, & Luo Y. (2014). Cyclin G2 Suppresses Estrogen-Mediated Osteogenesis through Inhibition of Wnt/β-Catenin Signaling. PLoS ONE, 9(3), e89884.
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7

Cell Lysis and Protein Detection

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Cells were lysed with RIPA buffer (Boston Bioproducts) supplemented with a cOmplete Mini EDTA-free Protease inhibitor cocktail (Roche) and a PhoSTOP phosphatase inhibitor cocktail (Roche). The total cell lysate (20 μg) was subjected to SDS polyacrylamide gel electrophoresis and transferred to Immobilon-P polyvinylidene difluoride membranes (Bio-Rad Laboratories). Antibodies used are listed in Supplementary Data 1.
Kobayashi Y., Oxnard G.R., Cohen E.F., Mahadevan N.R., Alessi J.V., Hung Y.P., Bertram A.A., Heppner D.E., Ribeiro M.F., Sacardo K.P., Saddi R., Macedo M.P., Blasco R.B., Li J., Kurppa K.J., Nguyen T., Voligny E., Ananda G., Chiarle R., Katz A., Tolstorukov M.Y., Sholl L.M, & Jänne P.A. (2022). Genomic and biological study of fusion genes as resistance mechanisms to EGFR inhibitors. Nature Communications, 13, 5614.
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8

Interrogating YAP, TEAD, and SLUG in EGFR-Mutant Cancer Cells

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PC-9 and HCC4006 cells treated for 48h with either DMSO or with the combination of 100nM osimertinib and 30nM trametinib were lysed in IP buffer (1% Triton-X100, 50mM Tris, 150mM NaCl, pH 7.4) supplemented with cOmplete Mini, EDTA-free Protease inhibitor cocktail (Roche) and PhoSTOP phosphatase inhibitor cocktail (Roche). 1500 μg of total protein was used for immunoprecipitation with Cell Signaling antibodies recognizing endogenous YAP (#14074), TEAD (pan-TEAD) (#13295), or SLUG (#9585) and 20 μl of Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology; sc-2003). Immunoprecipitations were carried out overnight in +4C, followed by four washes with 1 ml IP buffer. After washes, the beads were re-suspended in SDS Sample Buffer (Boston Bioproducts), and boiled for 5 min. Co-immunoprecipitated proteins were analyzed by western blotting.
Kurppa K.J., Liu Y., To C., Zhang T., Fan M., Vajdi A., Knelson E.H., Xie Y., Lim K., Cejas P., Portell A., Lizotte P.H., Ficarro S.B., Li S., Chen T., Haikala H.M., Wang H., Bahcall M., Gao Y., Shalhout S., Boettcher S., Shin B.H., Thai T., Wilkens M.K., Tillgren M.L., Mushajiang M., Xu M., Choi J., Bertram A.A., Ebert B.L., Beroukhim R., Bandopadhayay P., Awad M.M., Gokhale P.C., Kirschmeier P.T., Marto J.A., Camargo F.D., Haq R., Paweletz C.P., Wong K.K., Barbie D.A., Long H.W., Gray N.S, & Jänne P.A. (2020). Treatment-induced tumor dormancy through YAP-mediated transcriptional reprogramming of the apoptotic pathway. Cancer cell, 37(1), 104-122.e12.
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9

Protein Level Assessment in Dormant Cells

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If not specified below, cells were plated at 15 × 104 cells / cm2, treated the next day (if applicable) and lysed at specified timepoints in RIPA buffer (Boston Bioproducts) supplemented with cOmplete Mini EDTA-free Protease inhibitor cocktail (Roche) and PhoSTOP phosphatase inhibitor cocktail (Roche). Twenty micrograms of total protein was used for immunoblotting according to the antibody manufacturer’s recommendations. For the assessment of protein levels in dormant cells, 13 × 104 cells / cm2 were plated into 2 × 15 cm dishes. Cells were treated the next day and medium with fresh drugs was changed every 3–5 days. Cells were trypsinized at specified timepoints, washed with ice-cold PBS, and the cell pellets were lysed and immunoblotted as above.
Kurppa K.J., Liu Y., To C., Zhang T., Fan M., Vajdi A., Knelson E.H., Xie Y., Lim K., Cejas P., Portell A., Lizotte P.H., Ficarro S.B., Li S., Chen T., Haikala H.M., Wang H., Bahcall M., Gao Y., Shalhout S., Boettcher S., Shin B.H., Thai T., Wilkens M.K., Tillgren M.L., Mushajiang M., Xu M., Choi J., Bertram A.A., Ebert B.L., Beroukhim R., Bandopadhayay P., Awad M.M., Gokhale P.C., Kirschmeier P.T., Marto J.A., Camargo F.D., Haq R., Paweletz C.P., Wong K.K., Barbie D.A., Long H.W., Gray N.S, & Jänne P.A. (2020). Treatment-induced tumor dormancy through YAP-mediated transcriptional reprogramming of the apoptotic pathway. Cancer cell, 37(1), 104-122.e12.
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10

Quantitative Western Blot Analysis

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Protein was isolated from cultured cells by lysis in Triton X-100 lysis
buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA,
5 mM 2-mercaptoethanol, 10% glycerol, supplemented with Complete
protease inhibitor cocktail (Roche) and PhoSTOP phosphatase inhibitor cocktail
(Roche)). Protein was isolated from mouse tissue by grinding whole tissue in
Triton X-100 lysis buffer. Proteins were separated using SDS-PAGE and
transferred to PVDF membrane (Immobilon, Millpore, Billerica, MA, USA) for
western blotting. Secondary antibodies were detected using ECL (GE, Little
Chalfont, Buckinghamshire, UK), ECL 2 (Pierce, Rockford, IL, USA) or ECL Prime
(GE). Quantitation of ECL signal from p21WAF1/CIP1 western blots of
primary tissue was performed using ImageQuant (Molecular Dynamics, Little
Chalfont, Buckinghamshire, UK, version 5.2); signal was normalized to a loading
control (β actin or β tubulin) and expressed as normalized
signal relative to the normalized signal from the corresponding wild-type
control sample.
Dickerman B.K., White C.L., Kessler P.M., Sadler A.J., Williams B.R, & Sen G.C. (2015). The protein activator of protein kinase R, PACT/RAX, negatively regulates protein kinase R during mouse anterior pituitary development. The FEBS journal, 282(24), 4766-4781.
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