Phosphostop

Manufactured by Roche

Sourced in Germany, United States, Switzerland, France

PhosphoStop is a phosphatase inhibitor cocktail designed for the inhibition of serine/threonine and tyrosine phosphatases in biological samples. It is intended for use in the preparation of cell, tissue, or other biological samples prior to analysis of protein phosphorylation status.

Automatically generated - may contain errors

Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (20 mentions) Ripa buffer, by Thermo Fisher Scientific (18 mentions) Complete mini, by Roche (14 mentions) Complete protease inhibitor cocktail, by Roche (12 mentions) Ripa buffer, by Merck Group (11 mentions) Protease inhibitor cocktail, by Roche (9 mentions) Complete protease inhibitor, by Roche (8 mentions) Protease inhibitor, by Roche (7 mentions) Bradford assay, by Bio-Rad (6 mentions) β actin, by Cell Signaling Technology (4 mentions) Complete inhibitor cocktail, by Roche (4 mentions) Protein inhibitor cocktail, by Thermo Fisher Scientific (4 mentions) Image lab software, by Bio-Rad (4 mentions) Pvdf membrane, by Merck Group (4 mentions) Complete mini edta free, by Roche (4 mentions) Odyssey scanner, by LI COR (4 mentions) Dithiothreitol (dtt), by Merck Group (4 mentions) Bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Pvdf membrane, by Thermo Fisher Scientific (4 mentions) Gapdh, by Santa Cruz Biotechnology (3 mentions)

Close spelling variants of this product

Complete protease inhibitor and phosphoSTOP tablets PhosphoStop Inhibitors PhosphoSTOP™ EASYpacks phosphotase inhibitors cocktail PhosphoStop Phosphatase Inhibitor Cocktail PhosphoSTOP tablet Complete Protease Inhibitor and PhosphoStop Complete mini EDTA-free protease inhibitor cocktail and phosphostop PhosphoSTOP phosphatase inhibitor Protease inhibitor cocktail and phosphostop Complete and Phosphostop

148 protocols using phosphostop

Exosome Protein Isolation and Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were harvested and homogenized in RIPA lysis buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% TritonX-100, 0.1% SDS, 5 mM EDTA) containing complete protease inhibitor cocktail, phospho-stop (Roche Diagnostics, Meylan, France), DTT (Sigma-Aldrich) and vanadate (Thermo Fisher Scientific). Exosomes were centrifuged for 2 h at 100,000g at 4°C. The supernatants were discarded and the pellets were lysed by complemented RIPA lysis buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% TritonX-100, 0.1% SDS, 5 mM EDTA), phospho-stop, (Roche Diagnostics) and vanadate (Thermo Fisher Scientific). After 30 min on ice, protein content was measured by the BCA assay. Equal amounts of soluble protein (15–25 µg) were denatured by heating at 95°C for 5 min, resolved in SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% non-fat dry milk in TBS-Tween for 1 h and probed initially with specific primary antibody followed by horseradish peroxidase-conjugated secondary antibody. Blotted protein bands were detected by chemiluminescence (Supersignal, Thermo Fisher Scientific) exposure on X-ray films (Kodak, Rochester, NY, USA).

Ronquist K.G., Sanchez C., Dubois L., Chioureas D., Fonseca P., Larsson A., Ullén A., Yachnin J., Ronquist G, & Panaretakis T. (2016). Energy-requiring uptake of prostasomes and PC3 cell-derived exosomes into non-malignant and malignant cells. Journal of Extracellular Vesicles, 5, 10.3402/jev.v5.29877.

+ Open protocol

+ Expand

Immunoblotting Analysis of Cellular Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total cell lysates were prepared with 1% triton lysis buffer (25 mM Tris HCl (pH 8.0), 150 mM NaCl, 1% triton-X100, 1 mM dithiothreitol (DTT), protease inhibitor mix (Complete Mini, Roche) and phosphatase inhibitor (PhosphoStop, Roche)) and subjected to SDS-PAGE. The following antibodies were used for immunoblotting: anti-β-actin (Sigma-Aldrich), anti-cleaved PARP (Abcam, #ab32064), anti-PIP4K2A (#5527), anti-PIP4K2B (#9694), anti-cleaved caspase-3 (#9664), anti-pHistone H3(Ser10) (#3377), anti-pAkt(S473) (#9271), anti-pAkt(T308) (#13038), anti-total Akt (#9272), anti-p70S6K(T389) (#9234), anti-total p70S6K (#9202), anti-pErk (#4370), anti-γ-Histone H2AX (#9718) (Cell signaling), and anti-PIK3IP1 (Proteintech, #16826-1-AP). The secondary antibodies used were sheep anti-mouse IgG HRP and donkey anti-rabbit IgG HRP (Amersham; 1:2000 dilution). Immunoreactive proteins were visualized using ECL reagent (Amersham). Where indicated, intensities of protein bands were quantified by densitometry (Odyssey V3.0), normalized to their loading controls and then calculated as fold expression change relative to DMSO control. The uncropped scans of the blots were presented in Supplementary Fig. 9.

Kitagawa M., Liao P.J., Lee K.H., Wong J., Shang S.C., Minami N., Sampetrean O., Saya H., Lingyun D., Prabhu N., Diam G.K., Sobota R., Larsson A., Nordlund P., McCormick F., Ghosh S., Epstein D.M., Dymock B.W, & Lee S.H. (2017). Dual blockade of the lipid kinase PIP4Ks and mitotic pathways leads to cancer-selective lethality. Nature Communications, 8, 2200.

+ Open protocol

+ Expand

MAP Kinase In Vitro Kinase Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

The MAP kinase in vitro kinase assay was carried out as described by He et al., [48] (link). Briefly, 1 ml transfected protoplasts were lysed in 1 ml of immunoprecipitation (IP) buffer (150 mM NaCl, 50 mM HEPES pH 7.4, 1 mM EDTA, 1 mM DTT, 0,1% Triton X-100, 1× phosphatase inhibitor cocktail [PhosphoSTOP, Roche Applied Science] and 1× protease inhibitor cocktail [Complete EDTA-free, Roche Applied Science]). HA-tagged SlMPK1 and SlMPK3 kinases [52] (link) were immunoprecipitated from lysates after adding 20 µl anti-H antibody-coupled beads (Roche Applied Science) and incubated for 3 h at 4°C with gentle shaking. After centrifugation at 500 g for 1 min, the immunoprecipitated material was washed with IP buffer followed by a wash with kinase buffer (20 mM Tris-HCl pH 7.5, 20 mM MgCl₂, 5 mM EDTA and 1 mM DTT). The kinase reaction was performed by adding 25 µl of kinase buffer (0.25 mg/ml MBP, 100 µM ATP and 5 µCi [γ-³²P] ATP) for 30 min at RT. The reaction was stopped with 4× SDS-PAGE loading buffer. The ³²P-labeled MBP was separated by SDS/PAGE (15%) gel and visualized by autoradiography.

Zheng X., McLellan H., Fraiture M., Liu X., Boevink P.C., Gilroy E.M., Chen Y., Kandel K., Sessa G., Birch P.R, & Brunner F. (2014). Functionally Redundant RXLR Effectors from Phytophthora infestans Act at Different Steps to Suppress Early flg22-Triggered Immunity. PLoS Pathogens, 10(4), e1004057.

+ Open protocol

+ Expand

FLAG-tagged AKAP Protein Purification

Check if the same lab product or an alternative is used in the 5 most similar protocols

HEK293 cells were grown in Dulbecco’s Modified Eagle Medium or DMEM (GlutaMAX, 10 % FBS, Thermo Fisher Scientific) and transfected with the above-mentioned plasmids. The cells were lysed in lysis buffer (140 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 0.2 % Triton X-100, 2 mM EDTA, 2 mM EGTA) containing protease inhibitors (cOmplete, Roche) and phosphatase inhibitors (Phosphostop, Roche). The lysates were cleared by centrifugation (20 000 g, 4 °C, 10 min). Proteins were precipitated using anti-FLAG M2 magnetic beads (Sigma-Aldrich) [51 (link)] and eluted from the beads using 0.1 M glycine buffer, pH 2.5. The eluate was neutralized with 1 M Tris, pH 10.6, and incubated with Laemmli sample buffer for 8 min at 95 °C. Flag-tagged AKAPs, RIα, RIIα and RIIβ were detected by Western blotting with specific primary and horseradish peroxidase-coupled secondary antibodies (anti-Flag antibody: Sigma Aldrich #Flag M2; anti-RIα: BD Biosciences no. 610610; anti-RIIα: BD Biosciences no. 612243; anti-RIIβ: BD Biosciences no. 610626; anti-DDK (Flag): Origene TA50011, POD-anti-mouse IgG: Immuno Research no. 715-035-151) [10 (link),19 (link)].

Götz F., Roske Y., Schulz M.S., Autenrieth K., Bertinetti D., Faelber K., Zühlke K., Kreuchwig A., Kennedy E.J., Krause G., Daumke O., Herberg F.W., Heinemann U, & Klussmann E. (2016). AKAP18:PKA-RIIα structure reveals crucial anchor points for recognition of regulatory subunits of PKA. The Biochemical journal, 473(13), 1881-1894.

+ Open protocol

+ Expand

Western Blot Analysis of Key Cellular Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed using RIPA buffer (25 mM Tris.HCl, pH 7.6, 150 mM NaCl, 1% NP-40.0.25% sodium deoxycholate 0.1% SDS) supplemented with protease and phosphate inhibitor (PhosphoSTOP, Roche Diagnostics, Mannheim, Germany). Total Protein concentration was determined using a BCA assay kit (Ref 23,227, Pierce Thermo-Scientific, Rockford, IL, USA) and a spectrophotometer at 570 nm. Proteins were run on SDS-PAGE and transferred to nitrocellulose membrane for western blot analysis, using the appropriate antibodies. Primary antibodies: BRCA1 antibody (#9010 Cell Signaling), PARP1 (#9542 Cell Signaling), MDR1 (#12683 Cell Signaling), Bad Ser473 (#9271S Cell Signaling), Cleaved Caspase 3 (#9661 Cell Signaling), Bcl2 (#2876 Cell Signaling), p-Bad (#06–853 upstate Cell signaling), p-Bcl-2 (# 2875 Cell signaling) and β-actin (#4967 Cell Signaling). Secondary antibodies: anti-rabbit-HRP (L170–6515 Bio-Rad), anti-mouse HRP (L170–6516 Bio-Rad). Immunoblot proteins were visualized using horseradish peroxidase (HRP) conjugated secondary antibodies, and antigen-antibody complexes were detected using the ECL system.

Baloch T., López-Ozuna V.M., Wang Q., Matanis E., Kessous R., Kogan L., Yasmeen A, & Gotlieb W.H. (2019). Sequential therapeutic targeting of ovarian Cancer harboring dysfunctional BRCA1. BMC Cancer, 19, 44.

+ Open protocol

+ Expand

Sperm Capacitation and Tyrosine Phosphorylation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Aliquots of 5 × 10⁵ spermatozoa from wild-type and transgenic mice were incubated for various time points at 37°C in a humidified incubator with 5% CO₂ in noncapacitating and capacitating media. After incubation, sperm was centrifuged at 5 min at 10,000 rpm, and resuspended in lysis buffer (1 mM orthovanadate, 1 mM EDTA, 1 mM NaF, 1% NP-40, 1% deoxicolate, 0.1% SDS, and 1 × Phosphostop (Roche, Indianapolis, IN)). Samples were subjected to SDS-PAGE (7.5% gel) and immunoblotted, as previously described (Wagoner et al. 2005 (link)). Briefly, the membrane was blocked using 5% BSA in Tris-buffered saline with 0.5% Tween-20 (TBS-T) for 1 hour. Phosphorylation was detected by probing overnight at 4°C with anti-phosphotyrosine antibody (4G10 antibody; 1:1000 dilution in TBS-T Millipore, Billerica, MA. Blots were then wash 3 times with TBS-T, and then incubated for 1 hr with donkey anti-mouse IgG conjugated to horseradish peroxidase 1:1000 dilution in TBS-T. Following extensive washing, positive bands were detected using chemiluminescence. To ensure equal protein loading, membranes were stripped and re-probed with a polyclonal anti-α-tubulin antibody (1:1000 dilution) (Sigma-Aldrich). Phosphorylation intensities of the samples were analyzed with Pro-Gel Analyzer 4.5, and reported relative to the non-capacitated samples.

McDermott J., Sánchez G., Nangia A.K, & Blanco G. (2015). Role of Human Na,K-ATPase alpha 4 in Sperm Function, Derived from Studies in Transgenic Mice. Molecular reproduction and development, 82(3), 167-181.

+ Open protocol

+ Expand

Protein Extraction and Immunoblot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells and tissues were homogenized in Tissue Protein Extraction Reagent (Pierce) with EDTA, Complete protease inhibitor (Roche) and Phospho-STOP (Roche). Immunoblot analyses were performed as previously described (Foust et al., 2013 ). See Supplemental Experimental Procedures for details.

Frakes A.E., Ferraiuolo L., Haidet-Phillips A.M., Schmelzer L., Braun L., Miranda C.J., Ladner K.J., Bevan A.K., Foust K.D., Godbout J.P., Popovich P.G., Guttridge D.C, & Kaspar B.K. (2014). Microglia induce motor neuron death via the classical NF-κB pathway in amyotrophic lateral sclerosis. Neuron, 81(5), 1009-1023.

+ Open protocol

+ Expand

Western Blot Analysis of HMGCR Protein

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were washed with ice-cold PBS and lysed with ice-cold RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% 0.5% Na-deoxycholate, 0.1%sodium dodecyl sulfate, 10 mM TrisHCl pH 8) supplemented with phosphatase and protease inhibitor cocktails (Complete Mini and PhosphoStop, F. Hoffmann-La Roche Ltd). Lysates were centrifuged at 16,000g for 30 minutes at 4°C, supernatants were collected and the protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific Inc.). Equal amounts (20 μg) of protein were resolved on 4-12% Tris-HCL gels (Bio-Rad), and electrophoretically transferred to Immobilon-PVDF transfer membranes. Membranes were blocked for 1 hour in 5% non-fat milk in Tris-Buffered Saline (TBS)-Tween and then hybridized using a primary monoclonal antibody against HMGCR (AMAb90619, Atlas Antibodies, Sweden) at a dilution of 1:1000 in 5% non-fat milk TBS-Tween. β-actin was used as a loading control (1:1000, Cell Signalling Technology). Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000) were also prepared in 5% non-fat milk in TBS-Tween. Protein–antibody complexes were detected by chemiluminescence with the Clarity^™ ECL Western Blotting Substrate (Bio-Rad) and images were captured and the relative HMGCR protein expression was quantified with the CHEMIDOC^™ MP imaging system (Bio-Rad).

Kimbung S., Lettiero B., Feldt M., Bosch A, & Borgquist S. (2016). High expression of cholesterol biosynthesis genes is associated with resistance to statin treatment and inferior survival in breast cancer. Oncotarget, 7(37), 59640-59651.

+ Open protocol

+ Expand

Quantitative Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed with the RIPA lysis buffer (Thermo Fisher Scientific #89900) or in a buffer containing 1%NP40, 10mM EDTA, 150mM NaCl, 50mM Tris, with the protease inhibitor (cOmpleteTM mini protease inhibitor, Roche) and phosphatase inhibitor (phosphoSTOP, Roche) added freshly before the lysis. Using BCA Protein Assay Kit (Pierce), protein concentrations were measured, and equal amount of protein were loaded onto 4–12% Bis-Tris NuPAGE gels (Life Technologies). Gels were then transferred to nitrocellulose membranes, blocked with LI-COR TBS blocking buffer for 1 h, then probed with primary antibodies overnight. After washing three times in TBST (0.05% Tween in TBS), the secondary antibodies were added for 1hr rocking in RT, followed by three more washes in TBST. Blots were imaged using Odyssey Clx system (LI-COR) and quantified using ImageStudio (LICOR).

Lee H., Aylward A.J., Pearse RV I.I., Lish A.M., Hsieh Y.C., Augur Z.M., Benoit C.R., Chou V., Knupp A., Pan C., Goberdhan S., Duong D.M., Seyfried N.T., Bennett D.A., Taga M.F., Huynh K., Arnold M., Meikle P.J., Jager P.L., Menon V., Young J.E, & Young-Pearse T.L. (2023). Cell-type-specific regulation of APOE and CLU levels in human neurons by the Alzheimer’s disease risk gene SORL1. Cell reports, 42(8), 112994.

+ Open protocol

+ Expand

CAR T Cell Activation and Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

CAR.OTI cells were activated via either (i) solid phase antibodies or (ii) antigen-expressing tumor target cells. (i) CAR.OTI CTLs were cultured at 4 × 10⁶/mL with plate bound antibodies at 1 µg/mL; Armenian hamster anti-CD3e (clone 145-2C11; BD Bioscience), hamster anti-CD28 (clone 37-51; BD Bioscience), or mouse anti-c-myc Tag (clone 9B11; Cell Signaling). (ii) CAR.OTI CTLs and MC57 or MC57HER2 tumor target cells were incubated in serum-free media (±1 μM SIINFEKL peptide) for 1 h before coculturing 1:1 and incubating at 37 °C for various time points, before snap freezing (LN₂) in the presence of RIPA buffer (25 mmol/L Hepes, 0.25 mol/L NaCl, 2.5 mmol/L EDTA, and 0.1% Triton X-100), protease inhibitor mixture, and PhosphoStop (Roche Diagnostics). SDS PAGE followed by Western blot was then performed on the cell lysates using a 4–12% Bis-Tris gel (Invitrogen). PVDF membranes were probed with antibodies for 16 h at 4 °C, washed in PBS-T, and probed for 1 h at room temperature with HRP-conjugated secondary antibodies (anti-rabbit Ig or anti-mouse Ig; Dako), washed in PBS-T (3 × 5 min at room temperature) before digital imaging.

Davenport A.J., Cross R.S., Watson K.A., Liao Y., Shi W., Prince H.M., Beavis P.A., Trapani J.A., Kershaw M.H., Ritchie D.S., Darcy P.K., Neeson P.J, & Jenkins M.R. (2018). Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity. Proceedings of the National Academy of Sciences of the United States of America, 115(9), E2068-E2076.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!