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Phostop phosphatase inhibitor

Manufactured by Roche
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Sourced in Germany, Switzerland

PhoSTOP is a phosphatase inhibitor that helps researchers studying protein phosphorylation. It effectively inhibits a range of serine/threonine and tyrosine phosphatases.

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Lab products found in correlation

Ripa buffer, by Merck Group (2 mentions) Tissuelyser 2, by Qiagen (2 mentions) Complete mini protease inhibitor, by Roche (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Pvdf membrane, by Bio-Rad (2 mentions) Protein g sepharose, by Thermo Fisher Scientific (1 mentions) Ripa buffer, by Thermo Fisher Scientific (1 mentions) Sigmafast protease inhibitor cocktail, by Merck Group (1 mentions) Pro prep protein extraction solution, by iNtRON Biotechnology (1 mentions) Irdye 680lt goat anti rabbit igg, by LI COR (1 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions) Irdye 800cw goat anti mouse igg, by LI COR (1 mentions) Phospho rb ser780, by Cell Signaling Technology (1 mentions) Halt protease inhibitor, by Thermo Fisher Scientific (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions) Mcl 1, by Santa Cruz Biotechnology (1 mentions) Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibody, by Cell Signaling Technology (1 mentions) Mouse anti β actin, by Merck Group (1 mentions) Mes buffer, by Thermo Fisher Scientific (1 mentions) Pvdf membrane, by Merck Group (1 mentions)

13 protocols using phostop phosphatase inhibitor

1

Protein Immunoprecipitation and Western Blot

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Cells were suspended in lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 20 mM EDTA, 50 mM NaF, 0.5% NP-40, 1 mM dithiothreitol) supplemented with EDTA-free protease inhibitors and PhoSTOP phosphatase inhibitors (Roche, Mannheim, Germany). Lysates (500 μL) were then incubated with 2 μg antibody and 35 μL protein G–Sepharose (Invitrogen, Carlsbad, CA, USA) at 4°C overnight. Beads were washed 5 times with 1 mL lysis buffer before boiling in Laemmli sample buffer and performing SDS–polyacrylamide gel electrophoresis (PAGE), transfer to nitrocellulose membranes, and immunoblotting.
Boros K., Puissant A., Back M., Alexe G., Bassil C.F., Sinha P., Tholouli E., Stegmaier K., Byers R.J, & Rodig S.J. (2015). Increased SYK activity is associated with unfavorable outcome among patients with acute myeloid leukemia. Oncotarget, 6(28), 25575-25587.
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2

Western Blot Analysis Protocol

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Western blot analyses were performed as described previously33 (link). Briefly, cells were lysed using RIPA buffer (Invitrogen) containing SIGMAFAST Protease Inhibitor Cocktail (Sigma) and PhoSTOP phosphatase inhibitors (Roche). Adipose tissue lysates were prepared using PRO-PREP Protein Extraction Solution (iNtRON Biotechnology). Primary antibodies used for western blot analyses were described in Supplementary Tables S3. NIH ImageJ software was used for the quantification. Full-length immunoblot membrane images are attached in Supplementary Information.
Son Y., Choi C., Song C., Im H., Cho Y.K., Son J.S., Joo S., Joh Y., Lee Y.J., Seong J.K, & Lee Y.H. (2021). Development of CIDEA reporter mouse model and its application for screening thermogenic drugs. Scientific Reports, 11, 18429.
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3

Immunoblot Analysis of Palbociclib-Treated Tumor Tissue

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Mice bearing Med-211FH flank tumors were treated with palbociclib or vehicle. When tumors demonstrated evidence of regression, mice were humanely euthanized approximately four hours after the last dose. Tissue was preserved by snap freezing and formalin fixation. Tumor tissue was suspended in RIPA buffer (Millipore) containing Halt protease inhibitors (Pierce Biotechnology) and PhoStop phosphatase inhibitors (Roche) and lysed using the TissueLyser II (Qiagen). Equal amounts of protein (100 ug) were loaded and resolved by SDS-PAGE using Nu-PAGE 4–12% Bis-Tris gels (Invitrogen). Immunoblot was performed using standard techniques. Primary antibodies used were: Phospho-RB (Ser 780, #9307) and RB (#9309) (Cell Signaling Technology, 1:1000); and GAPDH (Santa Cruz Biotechnology, 1:500). Secondary antibodies used were: IRDye800CW Goat-anti-Mouse IgG and IRDye680LT Goat anti-Rabbit IgG (Li-Cor). Membranes were scanned using LiCor Odyssey. Image analysis was performed using Image Studio.
Cook Sangar M.L., Genovesi L.A., Nakamoto M.W., Davis M.J., Knobluagh S.E., Ji P., Millar A., Wainwright B.J, & Olson J.M. (2017). Inhibition of CDK4/6 by Palbociclib Significantly Extends Survival in Medulloblastoma Patient-Derived Xenograft Mouse Models. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(19), 5802-5813.
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4

Protein Expression Analysis in AML Cells

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Effect of DCA, ATO and their combination on different protein expression was measured in AML cell lines and primary AML cells. Total protein extracts were prepared using RIPA buffer (SIGMA) supplemented with Complete Mini™ protease inhibitor and PHOStop™ phosphatase inhibitors (Roche). Equal amounts of proteins (up to 25 μg) were separated on 4–12% NuPAGE gels in 1X MOPS or 1X MES buffer (Invitrogen) and transferred onto PVDF membranes (Millipore). The membrane was blocked with 5% dry milk in 1X TBS/0.1% Tween 20 (TBST) for at least one hour at room temperature, and incubated with human specific primary antibodies: GPx (Cell Signaling Technologies), and Mcl-1 (Santa Cruz) or mouse anti-β-actin (SIGMA) overnight at 4°C. Next day membrane was washed 3 times in TBST and incubated with a horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibody (Cell Signaling Technologies) for one hour at room temperature. Blots were again washed and the signal was detected with SuperSignal West Femto Chemiluminescent Substrate (Pierce) and exposed to HyBlot CL® autoradiography film (Denville). The signals were quantified using the Image J v.1.48s software (NIH). The signal for each antibody was normalized to β-actin and then relative difference was calculated against vehicle control at each time point (numbers below each Western blot strip).
Emadi A., Sadowska M., Carter-Cooper B., Bhatnagar V., van der Merwe I., Levis M.J., Sausville E.A, & Lapidus R.G. (2015). Perturbation of cellular oxidative state induced by dichloroacetate and arsenic trioxide for treatment of acute myeloid leukemia. Leukemia research, 39(7), 719-729.
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5

Cell Lysis and Phosphatase Inhibition

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Whole-cell lysates were generated using IGEPAL lysis buffer [50 mM tris (pH 7.4), 200 mM NaCl, 1% Igepal CA-630, 1 mM EDTA (pH 8.5)] containing sodium fluoride (1 mM), sodium vanadate (1 mM), protease inhibitor cocktail (Roche), phenylmethylsulfonyl fluoride (PMSF; 1 mM), 25 U of Pierce Universal Nuclease, 1 mM MgCl2, or PhoSTOP phosphatase inhibitors (Roche). Samples were analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The ATR inhibitor VX-970 was used at 1 μM where indicated. Samples were treated with lambda phosphatase (New England Biolabs, catalog no. P0753S; 400 U) for 30 min at 30°C.
Mehta K.P., Thada V., Zhao R., Krishnamoorthy A., Leser M., Lindsey Rose K, & Cortez D. (2022). CHK1 phosphorylates PRIMPOL to promote replication stress tolerance. Science Advances, 8(13), eabm0314.
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6

Comprehensive Cell Treatment Protocol

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Actinomycin D (A9415), α-Amanitin (A2263), Bovine Serum Albumin (BSA), 5-bromo-2′-deoxyuridine (BrdU, B5002), Cycloheximide, DAPI (4′,6-diamidino-2-phenylindole), Diethyl pyrocarbonate (DEPC), Dimethyl Sulfoxide (DMSO), Doxorubicin (D1515), 5-Fluorouracil (F6627), Leptomycin B (L2913), MG132 (C2211), Propidium iodide (PI), Sucrose, Tergitol/IGEPAL (NP-40), Triton™ X-100, Tween®20 were purchased from Sigma-Aldrich. Camptothecin (S1288) and Etoposide (S1225) were purchased from Selleck Chemicals and CX-5461 (SYN-3031) was obtained from SYNkinase. Dulbecco’s phosphate-buffered saline (dPBS, pH 7.4, Gibco) and ultrapure water (Invitrogen) were procured from ThermoFisher Scientific. 5X siRNA buffer (B-002000-UB-100, diluted to 1X with ultrapure water), DharmaFECT1 transfection reagent (T-2001) and assay-specific siRNAs (further details provided below) were purchased from Horizon Discovery. Polyethylenimine (PEI, #23966) was purchased from Polysciences. Paraformaldehyde (PFA, diluted in dPBS prior to use) was obtained from Electron Microscopy Sciences (Fisher Scientific). Complete protease inhibitor cocktail and PhoSTOP phosphatase inhibitor were purchased from Roche. All compounds used for treatment of cells, apart from α-Amanitin (H2O), CX-5461 (50 mM NaH2PO4, pH 4.5) and Leptomycin B (70% methanol) were solubilised in DMSO and stored at −20°C in aliquots prior to use.
Hannan K.M., Soo P., Wong M.S., Lee J.K., Hein N., Poh P., Wysoke K.D., Williams T.D., Montellese C., Smith L.K., Al-Obaidi S.J., Núñez-Villacís L., Pavy M., He J.S., Parsons K.M., Loring K.E., Morrison T., Diesch J., Burgio G., Ferreira R., Feng Z.P., Gould C.M., Madhamshettiwar P.B., Flygare J., Gonda T.J., Simpson K.J., Kutay U., Pearson R.B., Engel C., Watkins N.J., Hannan R.D, & George A.J. (2022). Nuclear stabilization of p53 requires a functional nucleolar surveillance pathway. Cell Reports, 41(5), 111571.
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7

Protein Extraction and Western Blot Analysis

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As described previously (Wang et al., 2017 (link)), the freshly colonic tissues were excised and fragmented using a scalpel. 500 ug–1 mg tissue portions were lysed for 30 min on ice with RIPA lysis buffer (Beyotime) in the presence of cocktail protease inhibitor (Roche) and phoSTOP phosphatase inhibitor (Roche). An additional step of sonication was also performed. Protein extracts (35 µg) were boiled and subjected to 10% SDS-PAGE gel before transfer to nitrocellulose membranes. The membranes were blocked for 2 h in PBST (PBS with 0.5% Tween 20) with 5% non-fat dry milk (Bio-Rad) and incubated with specific primary antibodies at 4°C overnight. Appropriate HRP-conjugated second antibodies (Cell Signaling Technology, #7076) were used at 1:3,000 dilution for 2 h at room temperature. Signals were detected by ECL HRP substrate (Advansta). The following primary antibodies were used: mouseβ-actin (Santa Cruz, sc-47778), mouse NF-κB p65 (Cell Signaling Technology, #6956), and mouse TNF-α (Abcam, ab1793).
Tang Z., Xiong D., Song J., Ye M., Liu J., Wang Z., Zhang L, & Xiao X. (2020). Antitumor Drug Combretastatin-A4 Phosphate Aggravates the Symptoms of Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice. Frontiers in Pharmacology, 11, 339.
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8

Quantification of Myelin Proteins in Mouse Spinal Cord

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To quantify protein amounts, 2-month-old mice were intracardially perfused with 1× PBS using a peristaltic pump (World Precision Instruments) for 3 min. Then, the spinal cord was removed and dissected to isolate the lumbosacral ventrolateral white matter, which was subsequently flash-frozen on dry ice. The tissue was lysed with fresh SDS lysis buffer with Mini Complete protease inhibitor and PhoStop phosphatase inhibitor (Roche) and homogenized with Precellys 24. A final protein concentration of 1 mg ml−1 was prepared in Laemmli buffer and 10 µg of protein was loaded per slot. The proteins were separated on a 12% Bis-Tris mini protein gel and run for 10 min at 80 V and 45 min at 200 V. Subsequently, proteins were transferred onto a PVDF membrane and detected using antibodies (MOG: Abcam, ab109746, rabbit, 1:2,000; secondary HRP-anti-rabbit, Cell Signaling, 7074 S, 1:15,000; MBP: Sigma, Atlas, AMAB91064, mouse, 1:5,000; secondary HRP-anti-mouse, Cell Signaling, 7076, 1:5,000; ACTB: Sigma, A5441, mouse, 1:2,500; secondary HRP-anti-mouse, Cell Signaling, 7076, 1:5,000). The antibodies were incubated separately and subsequently stripped using glycin, SDS and Tween for 10 min before proceeding with the next antibody. Detection was performed using ECL.
Schäffner E., Bosch-Queralt M., Edgar J.M., Lehning M., Strauß J., Fleischer N., Kungl T., Wieghofer P., Berghoff S.A., Reinert T., Krueger M., Morawski M., Möbius W., Barrantes-Freer A., Stieler J., Sun T., Saher G., Schwab M.H., Wrede C., Frosch M., Prinz M., Reich D.S., Flügel A., Stadelmann C., Fledrich R., Nave K.A, & Stassart R.M. (2023). Myelin insulation as a risk factor for axonal degeneration in autoimmune demyelinating disease. Nature Neuroscience, 26(7), 1218-1228.
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9

Ezrin and Cytoskeletal Protein Analysis in Stem Cells

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Harvested ES cells and EBs were lysed with RIPA lysis buffer (50mM Tris pH8.0, 150mM NaCl, 01% SDS, 0.5% Sodium deoxycholate, 1% NP40, complete protease inhibitor (Roche, 11697498001), and PhoSTOP phosphatase inhibitor (Roche, 04906845001)). A DC™ Protein assay reagent kit (BioRad, 500–0111) was used for measuring protein concentration. Equal protein amounts were separated by SDS/PAGE gel and transferred on PVDF membrane (BioRad, 1620177). The membranes were blocked and incubated overnight at 4oC with 1:50 anti-Ezrin (Abcam, ab4069), 1:1000 anti-alpha-Tubulin (Abcam, ab15246), 1:1000 anti-E-cadherin, 1:1000 anti- ß-catenin, and 1:1000 anti-LC3 (MBL, PD014). Antibody binding was detected by ECL. Protein quantification was analyzed with Image J.
Mah K., Soloff R., Izuhara A.K., Lakeland D.L., Wang C, & Mariani F.V. (2016). Prkci is required for a non-autonomous signal that coordinates cell polarity during cavitation. Developmental biology, 416(1), 82-97.
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10

Western Blot Analysis of Cell Signaling

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Western blotting was performed by lysing cells with sodium dodecyl sulfate (SDS) lysis buffer (1% SDS, 50mmol/L Tris-HCl pH 8.0, 10mmol/L EDTA) plus protease inhibitor cocktail (Sigma-Aldrich) and PhoSTOP phosphatase inhibitor (Roche) and sonication. Protein concentrations were standardized with a Micro BCA Protein Assay kit (Pierce Chemical), and each sample was homogenized in SDS sample buffer. SDS-polyacrylamide gel electrophoresis was performed using a 12% resolving gel. Wet transfer to PVDF membrane (Bio-Rad) was followed by blocking with 5% non-fat milk in TBS-T (TBS, 0.1% Tween-20). Incubation with primary antibodies was performed at a 1:500 dilution overnight at 4°C. Membranes were washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies at a 1:5000 dilution at room temperature for 1hr. Signal detection was performed on x-ray film using a chemiluminescence system (Amersham). Mouse primary antibody sources are: β-actin (MP Biomedicals), Bcl-2 (Santa Cruz), caspase-3 (Imgenex), γH2AX Ser139 (Millipore). Rabbit primary antibody sources are: H2AX (Millipore), p-p38 (Cell Signaling), PARP (Cell Signaling), c-myc (Cell Signaling), p38 (Cell Signaling).
Himmel L.E., Lustberg M.B., DeVries A.C., Poi M., Chen C.S, & Kulp S.K. (2016). Minocycline, a putative neuroprotectant, co-administered with doxorubicin-cyclophosphamide chemotherapy in a xenograft model of triple-negative breast cancer. Experimental and toxicologic pathology : official journal of the Gesellschaft fur Toxikologische Pathologie, 68(9), 505-515.
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