Phostop

Manufactured by Roche

Sourced in Switzerland, Germany, United States, United Kingdom

PhoSTOP is a phosphatase inhibitor cocktail designed for use in protein extraction and purification procedures. It effectively inhibits various classes of protein phosphatases, helping to preserve the phosphorylation state of proteins during sample preparation.

Automatically generated - may contain errors

Lab products found in correlation

Protease inhibitor cocktail, by Roche (10 mentions) Pvdf membrane, by Merck Group (10 mentions) Ripa buffer, by Merck Group (8 mentions) Bca protein assay kit, by Thermo Fisher Scientific (8 mentions) Complete mini, by Roche (6 mentions) Polyvinylidene fluoride (pvdf), by Merck Group (6 mentions) β actin, by Merck Group (5 mentions) Complete protease inhibitor, by Roche (4 mentions) Pierce ecl2, by GE Healthcare (4 mentions) Amersham imager 600, by GE Healthcare (4 mentions) Amersham ecl, by GE Healthcare (4 mentions) Ripa buffer, by Thermo Fisher Scientific (4 mentions) Odyssey infrared imaging system, by LI COR (3 mentions) Enhanced chemiluminescence detection system, by Bio-Rad (3 mentions) Complete protease inhibitor cocktail, by Roche (3 mentions) Bca assay, by Thermo Fisher Scientific (3 mentions) Cell lysis buffer, by Cell Signaling Technology (3 mentions) Pierce ecl, by Thermo Fisher Scientific (3 mentions) Clarity western ecl substrate, by Bio-Rad (2 mentions) Flag peptide, by Merck Group (2 mentions)

Close spelling variants of this product

Complete and PhoSTOP Complete PhoStop PhoSTOP phosphatase inhibitor cocktail PhoSTOP phosphatase inhibitor PhoSTOP EASY pack phosphatase inhibitor

88 protocols using phostop

Western Blot Analysis of Transfected Cells

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HEK293T cells were harvested 48 hours after transfection in Phosphate-Buffer Saline (PBS; Severn Biotech LTD) complemented with phosphatase inhibitors (PhoSTOP; Roche) and proteinase inhibitors (COMPLETE; Roche). LCLs were harvested by collecting cells in 15 mL tube (Falcon), centrifuged to form a pellet and resuspended in PBS complemented with phosphatase inhibitors (PhoSTOP; Roche) and proteinase inhibitors (COMPLETE; Roche). Cells were then lysed and processed as previously described (Scotter et al., 2014 (link)). Membrane imaging was conducted using goat anti-rabbit and anti-mouse IgG (H+L) DyLight 680 Conjugate (cat. no. 35568 and 35521, Thermo Life Sciences) and an LI-COR Odyssey or using horseradish peroxidase secondary antibodies for mice (Millipore, 12-349) or rabbits (Millipore, 12-348) and developed through an Enhanced Chemiluminescence System using Medical Film Processor SRX-101A (Konica Minolta). Western blot quantification was performed using the image analysis software, ImageJ (http://imagej.nih.gov/ij/(Schindelin et al., 2012 (link))).

de Majo M., Topp S.D., Smith B.N., Nishimura A.L., Chen H.J., Gkazi A.S., Miller J., Wong C.H., Vance C., Baas F., ten Asbroek A.L., Kenna K.P., Ticozzi N., Redondo A.G., Esteban-Pérez J., Tiloca C., Verde F., Duga S., Morrison K.E., Shaw P.J., Kirby J., Turner M.R., Talbot K., Hardiman O., Glass J.D., de Belleroche J., Gellera C., Ratti A., Al-Chalabi A., Brown R.H., Silani V., Landers J.E, & Shaw C.E. (2018). ALS-associated missense and nonsense TBK1 mutations can both cause loss of kinase function. Neurobiology of Aging, 71, 266.e1-266.e10.

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Western Blot Analysis of LDL Receptor

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Cultured cells were directly lysed for 15 min on ice with RIPA Lysis and Extraction Buffer (89900; Thermo Fisher Scientific Inc., Tokyo, Japan) containing with cOmplete™ ULTRA Tablets, Mini, EASYpack Protease Inhibitor Cocktail and PhoSTOP (05892970001 and 4906845001; Roche Diagnostics Co., Ltd., Tokyo, Japan). After centrifugation at 21,500 × g for 15 min, protein concentrations were measured using Pierce 660 nm Protein Assay Reagent (1861426; Thermo Fisher Scientific, Inc.), and protein was denatured by boiling for 5 min. Equal weights of protein (40 µg) protein was loaded onto sodium dodecyl sulfate-polyacrylamide gels for electrophoresis and then transferred onto nitrocellulose membranes. After blocking with 5% milk in TBST (150 mmol/l NaCl and 50 mmol/l Tris-HCl containing 0.05% Tween-20), the membranes were incubated with anti-LDL receptor antibody (dilution, 1:1,000) and anti-β-Actin antibody (dilution, 1:1,000) at 4°C overnight. After washing with TBST 3 times (5 min each), the membranes were incubated with their corresponding HRP-conjugated secondary antibodies (dilution, 1:5,000) at room temperature for 1 h. After washing with TBST 3 times (5 min each), bound antibodies were visualized using Clarity Western ECL Substrate (1705061; Bio-Rad Laboratories, Inc., Tokyo, Japan) and image analyzer (LAS-3000 mini; Fujifilm Co. Ltd., Tokyo, Japan).

Kanda T., Sugihara T., Takata T., Mae Y., Kinoshita H., Sakaguchi T., Hasegawa T., Kurumi H., Ikebuchi Y., Murakami T, & Isomoto H. (2019). Low-density lipoprotein receptor expression is involved in the beneficial effect of photodynamic therapy using talaporfin sodium on gastric cancer cells. Oncology Letters, 17(3), 3261-3266.

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Protein Extraction and Western Blot Analysis

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Cultured cells were either lysed with urea buffer (Fig 6E, 9 M urea and 20 mM Hepes–NaOH, pH 7.4) supplemented with 2-chloroacetamide (CAA; Sigma-Aldrich) or NP-40 (0.5% NP-40, 25 mM Tris–HCl, pH 7.5, 100 mM NaCl, and 50 mM NaF) lysis buffer and routinely supplemented with mammalian protease inhibitor (MPI) cocktail (Sigma-Aldrich) and Phostop (Roche), with the exception of data presented in Fig 2. Proteins were resolved using SDS–PAGE (Invitrogen NuPage gel 4–12%), transferred to nitrocellulose membrane, blocked in 5% milk, 5% BSA or 0.1% fish skin gelatin in TBS supplemented with Tween-20, and probed with primary antibodies overnight. Visualisation and quantification of Western blots were performed using IRdye 800CW and 680LT coupled secondary antibodies and an Odyssey infrared scanner (LI-COR Biosciences).

Rusilowicz-Jones E.V., Jardine J., Kallinos A., Pinto-Fernandez A., Guenther F., Giurrandino M., Barone F.G., McCarron K., Burke C.J., Murad A., Martinez A., Marcassa E., Gersch M., Buckmelter A.J., Kayser-Bricker K.J., Lamoliatte F., Gajbhiye A., Davis S., Scott H.C., Murphy E., England K., Mortiboys H., Komander D., Trost M., Kessler B.M., Ioannidis S., Ahlijanian M.K., Urbé S, & Clague M.J. (2020). USP30 sets a trigger threshold for PINK1–PARKIN amplification of mitochondrial ubiquitylation. Life Science Alliance, 3(8), e202000768.

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ChIP-seq and ChIP-qPCR with Spike-in Control

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ChIP-seq and ChIP-qPCR were performed as described previously (DeGennaro et al., 2013 (link)) with the following modifications. For the spike-in control, S. cerevisiae chromatin was added to S. pombe chromatin before immunoprecipitation with the relevant antibodies. S. cerevisiae chromatin corresponding to 2.5% (for early ChIP-seq) or 10% (for late ChIP-seq and ChIP-qPCR) of S. pombe chromatin, as estimated by Bradford assay (Bio-Rad), was used. For ChIP of Rpb1S2P and Rpb1S5P, phosphatase inhibitors (PhoSTOP, Roche) were added to the lysis buffer along with protease inhibitors (cOMPLETE tablet, Roche). Oligos for ChIP are listed in Table S2. ChIP-seq library prep was either as described previously (DeGennaro et al., 2013 (link)) or using the GeneRead DNA library prep kit (Qiagen) for end repair, A tailing and adapter ligation followed by two rounds of cleanup with 0.7 X SPRI beads (Beckman Coulter) for size selection. DNA was then amplified using an appropriate number of PCR cycles determined using a titration curve. The amplified DNA was subjected to two rounds of cleanup with 0.7 X SPRI beads. The DNA libraries were then processed as described previously (DeGennaro et al., 2013 (link)). All strains were done in duplicate. ChIP-seq libraries were sequenced on the Illumina HiSeq platform at the Tufts University or the NextSeq platform at the Harvard Bauer Core Facility.

Shetty A., Kallgren S.P., Demel C., Maier K.C., Spatt D., Alver B.H., Cramer P., Park P.J, & Winston F. (2017). Spt5 plays vital roles in the control of sense and antisense transcription elongation. Molecular cell, 66(1), 77-88.e5.

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Western Blot Analysis of Phospho-Src

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Neurons were lysed with ice-cold RIPA buffer (Sigma-Aldrich) supplemented with protease (Complete, Roche; 1 pill per 10 ml of lysis buffer) and phosphatase inhibitors (Phostop, Roche; 1 pill per 10 ml of lysis buffer). Lysates were centrifuged at 14,000 g for 10 min at 4 °C and supernatants were stored at −80 °C. Total protein concentration was determined by DC Kit (Bio-Rad Laboratories, Inc.) and 10 μg of total proteins were mixed with a same volume of sample buffer and incubated at 95 °C for 5 min and loaded into 10% PAGE gels (Mini-protean TGX precast gels, Bio-Rad Laboratories, Inc.). Proteins were transferred to PVDF membrane using Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Inc.). Blots were blocked for 30 min using Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA) and incubated with primary antibody for 2-hours at room temperature. The primary antibodies used were: rabbit anti-phospho Src-pY418 (Life Technologies, 1:1,000) and mouse anti-alpha tubulin (Abcam, 1:10,000). IRDye infrared secondary antibodies (LI-COR Biosciences) were used at concentration 1:10,000–1:20,000. Immunoreactivity was captured and quantified using the Odyssey Infrared imaging system (LI-COR Biosciences).

Berretta A., Gowing E.K., Jasoni C.L, & Clarkson A.N. (2016). Sonic hedgehog stimulates neurite outgrowth in a mechanical stretch model of reactive-astrogliosis. Scientific Reports, 6, 21896.

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Halo-TDP-43 i3Neurons Protein Analysis

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Halo-TDP-43 i3Neurons were homogenised in lysis buffer (25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% Glycerol, 2 mM EDTA, 0,1% SDS, protease inhibitor (cOmplete^™ EDTA-free protease inhibitor cocktail, Roche), phosphatase inhibitor (PhoSTOP^™, Roche)). Samples were loaded on a NuPAGE 4–12% Bis-Tris protein gel (Invitrogen), which was run in NuPAGE MOPS buffer. Proteins were transferred onto PVDF blotting membrane (Amersham), through wet transfer for 1h 30 min at 200 mA in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). The membrane was blocked in 5% milk in TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween-20) and incubated overnight with primary antibodies diluted in 5% milk in TBST (anti-ELK1 (Abcam ab32106) 1:500, anti-TDP-43 (Abcam, ab104223) 1:2000, anti-tubulin (Sigma-Aldrich, MAB1637) 1:5000). After 1h-incubation with HRP-conjugated secondary antibodies diluted in 5% milk in TBST (anti-mouse HRP (BioRad, 1706516) 1:10000, anti-rabbit HRP (BioRad 1706515) 1:10000), the membrane was developed using Immobilon Classico HRP substrate (Sigma) and the Bio-Rad ChemiDoc system.

Bryce-Smith S., Brown A.L., Mehta P.R., Mattedi F., Mikheenko A., Barattucci S., Zanovello M., Dattilo D., Yome M., Hill S.E., Qi Y.A., Wilkins O.G., Sun K., Ryadnov E., Wan Y., Vargas J.N., Birsa N., Raj T., Humphrey J., Keuss M., Ward M., Secrier M, & Fratta P. (2024). TDP-43 loss induces extensive cryptic polyadenylation in ALS/FTD. bioRxiv.

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Quantitative Protein Analysis of Rabbit Mammary Gland

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Protein extracts from each rabbit mammary gland were prepared in 50 mM Tris‐HCl (pH 8), 137 mM NaCl, 2.7 mM KCl, 1% NP40, and 10% glycerol, containing protease and phosphatase inhibitors (Complete Mini and PhoSTOP, Roche, Meylan, France). Their protein concentration was determined using the BCA Protein Assay kit (Thermo Scientific, France). Proteins (5 μg) were separated on 10% SDS‐polyacrylamide gel and transferred onto nitrocellulose filters (Bio‐rad, France). The membranes were saturated for 1 hour in blocking solution (TBS‐T:50 mM Tris pH 7.4, 200 mM NaCl, 0.1% Tween 20, 5% defatted milk) and incubated overnight with the primary antibodies at 4°C. After incubation, the membranes were washed with TBS‐T and incubated at room temperature with horseradish peroxidase‐linked secondary antibody for 45 minutes. Immune complexes were detected using the ECL kit for autoradiography (GE Healthcare, France). The following specific antibodies were used: guinea pig anti‐rabbit Wap (1:5000) antibodies,68 and anti‐β‐actin mouse antibody (1:10 000, Clone AC‐15, Sigma‐Aldrich, France) as loading control. The secondary antibodies used for immunoblotting were: anti‐guinea pig (1:5000) and anti‐mouse (1:10 000) horseradish peroxidase (HRP)‐conjugated (Sigma‐Aldrich, France). Quantification was assayed using ImageJ software and results are expressed as means ± SEM.

Hue‐Beauvais C., Laubier J., Brun N., Houtia I., Jaffrezic F., Bevilacqua C., Le Provost F, & Charlier M. (2019). Puberty is a critical window for the impact of diet on mammary gland development in the rabbit. Developmental Dynamics, 248(10), 948-960.

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Western Blot Analysis of Protein Lysates

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Western blot analysis was performed as previously described with minor modification^{36 (link)}. Cells were lysed with RIPA buffer (Sigma Aldrich, St. Louis, MO, USA) containing a protease inhibitor cocktail and PhoSTOP (Roche Molecular Biochemicals, Basel, Switzerland). Ten micrograms of the protein lysate were loaded into 10% SDS-PAGE and transferred to PVDF membranes (Amersham, Arlington Heights, IL, USA). After transfer is finished, the membranes were incubated with blocking solution and then appropriate antibodies. The bands were visualized with Advanced ECL Western Blotting Detection Reagents (Amersham). GAPDH levels are used as a loading control.

Lee M.H., Lee Y.S., Kim H.J., Han C.H., Kang S.U, & Kim C.H. (2019). Non-thermal plasma inhibits mast cell activation and ameliorates allergic skin inflammatory diseases in NC/Nga mice. Scientific Reports, 9, 13510.

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Myeloperoxidase Quantification Protocol

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Samples were homogenized in lysis buffer (20 mM Tris-HCl pH 7.5, 250 mM succrose, 20 mM ETDA, 3 mM EGTA, 0.1% Triton X-100, supplemented with 10× ETDA-free Protease Inhibitor Tablets and 10× PhoSTOP; Roche Diagnostics) using the Tissue Lyzer (Qiagen). Homogenates were centrifuged at 14,000 g (4°C, 10 min) and the supernatant was recovered. MPO was quantified using an ELISA (Hycult Biotechnology) according to the manufacture's instruction. Total protein amount in samples was assessed with BCA-protein assay (Pierce). MPO levels were related to total protein.

Friedrichs K., Adam M., Remane L., Mollenhauer M., Rudolph V., Rudolph T.K., Andrié R.P., Stöckigt F., Schrickel J.W., Ravekes T., Deuschl F., Nickenig G., Willems S., Baldus S, & Klinke A. (2014). Induction of Atrial Fibrillation by Neutrophils Critically Depends on CD11b/CD18 Integrins. PLoS ONE, 9(2), e89307.

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Immunoprecipitation of Mnat9 in Drosophila S2 Cells

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A Midi-prep kit (Macherey-Nagel) was used to prepare high-purity (A260/280 ~1.8) plasmid DNA. For transfection, Drosophila S2 cells were mixed with 1–2 μg of plasmid DNA and Effectene (Qiagen) following the manufacturer’s instructions. After 48 h, cells were harvested and lysed using a glass homogenizer with HEPES buffer (0.5% CHAPS) at 4 °C. To prevent protein degradation or dephosphorylation, protease-inhibitor cocktail (Roche) and PHOSTOP (Roche) were used. Samples were centrifuged at 13,000 g at 4 °C for 15 min, and the supernatant was used for further experiments. Beads (Surebead, Bio-rad) were precleared for 30 min at RT. Precleared beads were incubated with control rabbit IgG (Genscript-A01008) or Mnat9 antibody (1:100) [21 ] for 1 h at 4 °C. Antibody-bound beads were incubated with samples for 2 h at 4 °C and washed at least four times before elution.

Mok J.W, & Choi K.W. (2022). Modulation of Hippo signaling by Mnat9 N-acetyltransferase for normal growth and tumorigenesis in Drosophila. Cell Death & Disease, 13(2), 101.

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