Phosstop inhibitor

Manufactured by Roche

Sourced in Canada, Switzerland

PhosSTOP is a ready-to-use phosphatase inhibitor cocktail that can be added directly to cell lysates or protein samples to inhibit the activity of serine/threonine and tyrosine phosphatases. It is designed to prevent dephosphorylation of proteins during sample preparation and processing.

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Lab products found in correlation

Complete protease inhibitor, by Roche (2 mentions) Rapigest sf, by Waters Corporation (2 mentions) Bradford assay, by Bio-Rad (2 mentions) Protease inhibitor cocktail tablet, by Roche (2 mentions) Amersham protran membrane, by GE Healthcare (1 mentions) Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions) Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Qiashredder spin column, by Qiagen (1 mentions) Dnase digestion, by Qiagen (1 mentions) Teabc, by Merck Group (1 mentions) Rneasy mini column, by Qiagen (1 mentions) Qiazol lysis reagent, by Qiagen (1 mentions) Immobilon pvdf membrane, by Merck Group (1 mentions) Memcode, by Thermo Fisher Scientific (1 mentions) Cellytic m cell lysis reagent, by Merck Group (1 mentions) C digit blot scanner, by LI COR (1 mentions) Image studio, by LI COR (1 mentions) Imagequant las 4000, by GE Healthcare (1 mentions) Mineral oil, by Merck Group (1 mentions) Favorprep tissue genomic dna extraction mini kit, by Favorgen Biotech (1 mentions)

11 protocols using phosstop inhibitor

Cell Lysis and Protein Analysis

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For whole-cell lysates, cells were obtained using non-denaturing lysis buffer (NDLB) (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 5 mM EDTA, and 1% Triton X-100 (w/v) supplemented with 1 mM PMSF, 1× PhosSTOP inhibitor (Roche), and 10 mM NaF). Samples were gently agitated at 4 °C for 30 min, followed by clarification by spinning in a microfuge at 13,000 RPM for 10 min at 4 °C. For cytoplasmic fractions, cells were lysed using 10 mM HEPES pH 7.8, 10 mM KCl, 2 mM MgCl₂, 0.1 mM EDTA, 1 mM PMSF, 1× PhosSTOP inhibitor (Roche), and 10 mM NaF, and incubated on ice for 30 min. Lysates were vortexed, sheared 8 times with a 25 G needle, and clarified by spinning in a microfuge at 13,000 RPM for 5 min at 4 °C. Proteins were analyzed by SDS-PAGE and immunoblotting. Chemiluminescence signal was detected using a C-DiGit blot scanner (LI-COR). Obtained images and band intensity were analyzed using Image Studio (LI-COR).

Ng V.H., Spencer Z., Neitzel L.R., Nayak A., Loberg M.A., Shen C., Kassel S.N., Kroh H.K., An Z., Anthony C.C., Bryant J.M., Lawson A., Goldsmith L., Benchabane H., Hansen A.G., Li J., D’Souza S., Lebensohn A.M., Rohatgi R., Weiss W.A., Weiss V.L., Williams C., Hong C.C., Robbins D.J., Ahmed Y, & Lee E. (2023). The USP46 complex deubiquitylates LRP6 to promote Wnt/β-catenin signaling. Nature Communications, 14, 6173.

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Protein Isolation and Western Blot Analysis

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In order to isolate protein, spheroids were harvested with RIPA Lysis and Extraction buffer (Thermo Fisher Scientific) supplemented with cOmplete protease and PhosSTOP inhibitors (Roche). Aliquots of the protein homogenate were subjected to SDS-PAGE and Western blot analysis according to Karlgren et al.^{26 (link)} with minor modifications. AmershamProtran membrane (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) was used and visualization was performed using the Super Signal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Waltham, MA USA). Primary antibodies for GSK3β (#9832 CellSignal) and Phospho-GSK-3β (#9336 CellSignal) were used.

Kozyra M., Johansson I., Nordling Å., Ullah S., Lauschke V.M, & Ingelman-Sundberg M. (2018). Human hepatic 3D spheroids as a model for steatosis and insulin resistance. Scientific Reports, 8, 14297.

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Co-immunoprecipitation and GST Pulldown of XIAP-RIPK2

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For co‐immunoprecipitation of HA‐XIAP with RIPK2, transfected and treated cells were lysed in TBS buffer containing 0.5% NP‐40, cOmplete and PhosSTOP inhibitors (Roche) and the lysate was cleared by centrifugation. The lysates were incubated with anti‐HA‐agarose overnight, washed 3× with lysis buffer, and analyzed by immunoblotting. For GST pulldown experiments, U2OS/NOD2 cells were lysed in TBS lysis buffer containing 0.5% NP‐40, cOmplete and PhosSTOP inhibitors (Roche). Cleared lysates were pretreated with kinase inhibitors or DMSO and incubated with GST‐XIAP‐BIR2 bound to Glutathione Sepharose at 4°C overnight. Bound material was washed 3× with lysis buffer or PBS, eluted with 15 mM Glutathione in PBS, and analyzed by immunoblotting. In pulldown experiments with recombinant (dephosphorylated) RIPK2 kinase domain (Canning et al, 2015), inhibitors or DMSO control in PBS were added together with RIPK2 kinase domain to GST‐XIAP‐BIR2 bound to Glutathione Sepharose. The bound material was eluted and analyzed as described above. Ubiquitin conjugates were purified from treated cells using GST‐1xUBA^ubq ubiquitin affinity reagent (termed TUBE pulldown) and analyzed by immunoblotting as described (Fiil et al, 2013).

Hrdinka M., Schlicher L., Dai B., Pinkas D.M., Bufton J.C., Picaud S., Ward J.A., Rogers C., Suebsuwong C., Nikhar S., Cuny G.D., Huber K.V., Filippakopoulos P., Bullock A.N., Degterev A, & Gyrd‐Hansen M. (2018). Small molecule inhibitors reveal an indispensable scaffolding role of RIPK2 in NOD2 signaling. The EMBO Journal, 37(17), e99372.

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Protein Kinase Inhibitor Dose Response

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SCC12 cells were plated at 5 × 10⁵per well of a 6 well plate. After 24 hours, medium was replaced with medium supplemented with DMSO or a dose range of BDP9066. After 2 hours, cells were washed in PBS and lysed in 1% (w/v) SDS, 50 mM Tris-HCl, pH 7.4. Lysates were passed through QIAshredder spin columns (79654; Qiagen) and analyzed by SDS-PAGE.
HEK293 cells were transfected with pEF-FLAG-MRCKα. After 48 hours, medium was replaced with medium supplemented with either DMSO, 3 μM BDP5290 or 1 μM BDP9066. After 2 hours, cells were washed in PBS and lysed in 1% (w/v) SDS, 50 mM Tris-HCL pH 7.4. Lysates were passed through QIAshredder spin columns and analyzed by SDS-PAGE.
MDA MB 231 cells expressing doxycycline inducible ROCK1, ROCK2 or MRCKβ kinase domains were plated at 1.1 x 10⁵ per well of a 12 well plate. After 24 hours, cells were treated with 1 µg/ml doxycycline for 18 hours to induce kinase domain expression and then tested with BDP8900 or BDP9066 at the concentrations indicated for 60 minutes. Cells were then washed with PBS and lysed with Tris-SDS lysis Buffer (50 mM Tris-HCl Ph 7.4, 0.5% (v/v) SDS, 1x PhosStop Inhibitors (04 906 837 001; Roche) and 1x Complete Protease Inhibitors (04 693 124 001; Roche). Whole cell lysates were clarified by passing through QIAshredder spin columns and analyzed by SDS-PAGE.

Unbekandt M., Belshaw S., Bower J., Clarke M., Cordes J., Crighton D., Croft D.R., Drysdale M.J., Garnett M.J., Gill K., Gray C., Greenhalgh D.A., Hall J.A., Konczal J., Lilla S., McArthur D., McConnell P., McDonald L., McGarry L., McKinnon H., McMenemy C., Mezna M., Morrice N.A., Munro J., Naylor G., Rath N., Schüttelkopf A.W., Sime M, & Olson M.F. (2018). Discovery of potent and selective MRCK inhibitors with therapeutic effect on skin cancer. Cancer research, 78(8), 2096-2114.

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SARS-CoV-2 Infection Dynamics in Calu-3 Cells

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Calu-3 cells were grown in complete DMEM-F12 media in a humidified incubator at 37 °C in the presence of 5% CO2. The medium was replaced with DMEM-F12 containing 0.2% BSA and Pen-Strep prior to infection experiments. The cells were mock- or virus-infected at an MOI of 0.1 for 0, 3, 6, 12, 24, and 48 h. At each time point, the samples were washed twice with 1x TBS buffer and harvested in QIAzol lysis reagent (Qiagen, Germany) (for RNASeq) followed by DNAse digestion (Qiagen, Germany) treatment on the RNeasy Mini columns, according to the manufacturer’s protocol. SDS lysis buffer 4%SDS, 50 mM TEABC (Sigma Aldrich), and PhosStop inhibitors (Roche) was used to extract the proteins. The lysates for proteomic and PTMomic analysis were heat-inactivated at 90°C for 10 min and stored at −80°C until further processing.

Pinto S.M., Subbannayya Y., Kim H., Hagen L., Górna M.W., Nieminen A.I., Bjørås M., Espevik T., Kainov D, & Kandasamy R.K. (2022). Multi-OMICs landscape of SARS-CoV-2-induced host responses in human lung epithelial cells. iScience, 26(1), 105895.

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Western Blot Analysis of Cardiomyocytes

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For Western blots, cardiomyocytes were collected from plates and lysed using a CelLytic M Cell Lysis Reagent (Sigma Aldrich, Canada) with cOmplete and PhosSTOP inhibitors (Roche, Canada). Upon transfer to Immobilon PVDF membranes (EMD Millipore, Canada), antibodies used were from Cell Signaling (Product ID: 9272, 9271, 9275 and 7074). PVDF membranes were stained for total protein as a loading control using MemCode (Thermo Fisher Scientific).

Zhabyeyev P., McLean B., Chen X., Vanhaesebroeck B, & Oudit G.Y. (2019). Inhibition of PI3Kinase-α is pro-arrhythmic and associated with enhanced late Na⁺ current, contractility, and Ca²⁺ release in murine hearts. Journal of molecular and cellular cardiology, 132.

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Muscle Protein Expression Analysis

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Total RNA preparation and quantitative real-time PCR were performed as described [22 (link)]. Expression was normalized to 36b4 expression. Primer sequences are as previously described [29 (link)]. Frozen muscle was homogenized in ice-cold Tris-Triton buffer (10mM Tris, pH 7.4, 100mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, and 0.5% deoxycholate) with 1mM PMSF, 1X cOmplete protease inhibitor and PhosSTOP inhibitor from Roche) with a motorized Teflon pestle. Primary and secondary antibodies used were described previously [29 (link)]. Antibody binding was detected by ECL Plus (Pierce), using ImageQuant LAS4000 (GE) for image capture. Fiji was used for quantification of band intensity.

Cortez-Toledo O., Schnair C., Sangngern P., Metzger D, & Chao L.C. (2017). Nur77 deletion impairs muscle growth during developmental myogenesis and muscle regeneration in mice. PLoS ONE, 12(2), e0171268.

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Comprehensive Molecular Analysis Techniques

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Total RNA was extracted using a FavorPrepTM Blood/Cultured Cell Total RNA Mini Kit (Favorgen Biotech, China). Genomic DNA was prepared by using a FavorPrep Tissue Genomic DNA Extraction Mini Kit (Favorgen Biotech, China). For Western blotting, signals were visualized by using a SuperSignal West Femto Maximum Sensitivity Substrate Kit (Thermo, USA). Lymphocyte isolation was performed with a Ficoll density gradient centrifugation kit (GE Healthcare, USA). Cell-free DNA was isolated and quantified by using a Dynabeads® SILANE Viral NA Kit (Thermo, USA) and Quant-iT™ PicoGreen ® dsDNA Reagent and Kits (Thermo, USA).
Reagents and services: LipofectamineTM 3000 (Thermo, USA); TREX1 siRNA (Santa Cruz, USA); cGAS siRNA (Santa Cruz, USA); siRNA control plasmid (Sigma, USA); Mycobacterium tuberculosis (M. tuberculosis; Difco, USA); mineral oil (Sigma, USA); methotrexate (MTX) (LC labs, USA); Cre adeno-associated virus construction (Ubigene, China); TREX1 adeno-associated virus construction (Ubigene, China); dimethyl sulfoxide (DMSO; ACROS, USA); DAPI (Invitrogen, USA); RIPA buffer (10×, Cell Signaling Technology, USA); EDTA-free protease inhibitors, PhosSTOP inhibitor (Roche, Basel, Switzerland); TRIzol (Invitrogen, USA); FastStart Universal SYBR Green Master Rox (Roche Diagnostics, USA); Maxima™ H Minus cDNA Synthesis Master Mix (Thermo, USA); PVDF membranes (Bio-Rad, USA).

Luo W.D., Wang Y.P., Lv J., Liu Y., Qu Y.Q., Xu X.F., Yang L.J., Lin Z.C., Wang L.N., Chen R.H., Yang J.J., Zeng Y.L., Zhang R.L., Huang B.X., Yun X.Y., Wang X.Y., Song L.L., Wu J.H., Wang X.X., Chen X., Zhang W., Wang H.M., Qu L.Q., Liu M.H., Liu L., Law B.Y, & Wong V.K. (2023). Age-related self-DNA accumulation may accelerate arthritis in rats and in human rheumatoid arthritis. Nature Communications, 14, 4394.

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Cellular Lysis and Protein Extraction

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For immunoblotting, cells were lysed in 1% (v/v) NP-40, 1% (w/v) SDS dissolved in 50 mM Tris–HCl pH 8.0 and 150 mM NaCl, supplemented with Roche protease inhibitor cocktail tablet. The lysate was sonicated briefly and centrifuged at 15 000×g (4°C) for 20 min. Protein concentration was quantified using the Bradford Assay (Bio-Rad). For co-immunoprecipitation (IP) experiments, cells were lysed in 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.5% NP-40, 1 mM DTT, 2 mM MgCl₂, and benzonase supplemented with a protease inhibitor (Roche) cocktail tablet. After 30 min incubation on ice, cell lysates were clarified by centrifugation (15 000×g, 4°C, 20 min). For LC–MS/MS analysis, cells were lysed with an MS compatible buffer (0.25% (v/v) RapiGest SF (Waters) in 50 mM ammonium bicarbonate, supplemented with PhosStop inhibitor (Roche). Lysates were sonicated briefly to shear DNA and centrifuged (15 000×g, 4°C, 20 min).

Byrne D.P., Clarke C.J., Brownridge P.J., Kalyuzhnyy A., Perkins S., Campbell A., Mason D., Jones A.R., Eyers P.A, & Eyers C.E. (2020). Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signalling networks using SILAC-based phosphoproteomics. Biochemical Journal, 477(13), 2451-2475.

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Protein Extraction and Analysis Protocols

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For immunoblotting, cells were lysed in 1% (v/v) NP-40, 1% (w/v) SDS dissolved in 50 mM Tris-HCl pH 8.0 and 150 mM NaCl, supplemented with Roche protease inhibitor cocktail tablet.
The lysate was sonicated briefly and centrifuged at 15,000 xg (4 °C) for 20 min. Protein concentration was quantified using the Bradford Assay (BioRad). For co-immunoprecipitation (IP) experiments, cells were lysed in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP-40, 1 mM DTT, 2 mM MgCl2, and benzonase supplemented with a protease inhibitor (Roche) cocktail tablet. After 30 min incubation on ice, cell lysates were clarified by centrifugation (15,000 xg, 4 °C, 20 min). For LC-MS/MS analysis, cells were lysed with an MS compatible buffer (0.25% (v/v) RapiGest SF (Waters) in 50 mM ammonium bicarbonate, supplemented with PhosStop inhibitor (Roche). Lysates were sonicated briefly to shear DNA and centrifuged (15,000 xg, 4 °C, 20 min).

Byrne D.P., Clarke C.J., Brownridge P., Kalyuzhnyy A., Perkins S., Campbell A., Mason D.Y., Jones A.R., Eyers P.A., & Eyers C.E. (2020). Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signaling networks using SILAC-based phosphoproteomics.

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