Phosstop phosphatase inhibitor cocktail

Manufactured by Roche

Sourced in Germany, Switzerland, United States, Japan, China, Sweden, France, United Kingdom, Italy

PhosSTOP is a phosphatase inhibitor cocktail. It is designed to inhibit a broad range of serine/threonine and tyrosine phosphatases, thereby preventing the dephosphorylation of phosphoproteins during sample preparation and analysis.

Automatically generated - may contain errors

Lab products found in correlation

Complete protease inhibitor cocktail, by Roche (119 mentions) Protease inhibitor cocktail, by Roche (35 mentions) Pvdf membrane, by Merck Group (31 mentions) Protease inhibitor cocktail, by Merck Group (28 mentions) Complete edta free protease inhibitor cocktail, by Roche (24 mentions) Complete mini protease inhibitor cocktail, by Roche (22 mentions) Pvdf membrane, by Bio-Rad (14 mentions) Ripa buffer, by Thermo Fisher Scientific (13 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (12 mentions) β actin, by Merck Group (11 mentions) Protease inhibitor cocktail, by Nacalai Tesque (10 mentions) Ripa buffer, by Merck Group (10 mentions) Complete protease inhibitor, by Roche (9 mentions) Gapdh, by Santa Cruz Biotechnology (8 mentions) Bca kit, by Thermo Fisher Scientific (7 mentions) β actin, by Cell Signaling Technology (7 mentions) Anti β actin, by Merck Group (6 mentions) Las 4000, by Cytiva (6 mentions) Cell lysis buffer, by Cell Signaling Technology (6 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)

Close spelling variants of this product

PhosSTOP (1722 citations) Phosphatase inhibitors (745 citations) Phosphatase inhibitor cocktail (633 citations) PhosSTOP phosphatase inhibitor (356 citations) PhosSTOP cocktail (19 citations) PhosSTOP inhibitor (11 citations) PhosStop inhibitor cocktail (7 citations) Cocktail inhibitor (7 citations) Inhibitor cocktails (2 citations) Phosphatases (2 citations)

409 protocols using phosstop phosphatase inhibitor cocktail

Fractionation and Quantification of Tau

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

To measure the levels of tau and hyperphosphorylated tau epitopes, soluble (in reassembly buffer (RAB)) and insoluble (RIPA) fractions of hippocampal homogenate were sequentially extracted as previously described (Probst et al., 2000 (link)). Briefly, hippocampi were suspended in 10 volumes/weight ice-cold RAB buffer (0.01 M MES, 1 mM EGTA, 0.5 mM MgSO₄, 0.75 M NaCl, 0.02 M NaF, 1 mM Na₃VO₄, 1 mM PMSF) containing Complete EDTA-free Protease Inhibitor Cocktail (Roche) and PhosSTOP Phosphatase Inhibitor Cocktail (Roche), and homogenized. Samples were centrifuged at 21,000 g for 90 min at 4°C and the supernatant was extracted as the RAB fraction. The remaining pellet was resuspended and homogenized in the same volume of ice-cold RIPA buffer (Cell Signaling) with 0.02 M NaF, 1 mM Na₃VO₄, 1 mM PMSF, Complete EDTA-free Protease Inhibitor Cocktail (Roche) and PhosSTOP Phosphatase Inhibitor Cocktail (Roche). Samples were centrifuged at 21,000 g for 90 min at 4°C and the supernatant was extracted as the RIPA fraction. A BCA assay (Thermo Scientific) was performed in order to measure the total protein content for each sample from each fraction to ensure that changes in tau were comparable across samples.

Turnbull M.T., Boskovic Z, & Coulson E.J. (2018). Acute Down-regulation of BDNF Signaling Does Not Replicate Exacerbated Amyloid-β Levels and Cognitive Impairment Induced by Cholinergic Basal Forebrain Lesion. Frontiers in Molecular Neuroscience, 11, 51.

+ Open protocol

+ Expand

Extraction and Quantification of Proteins from Cell Lines and Mouse Tissues

Cited 1 time

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

Cell culture media from HEK-293 cells were collected and cleared from insoluble material by centrifugation for 10 min at 16,000×g, 4 °C. Cells were washed with PBS and extracts were prepared by scraping and vortexing the cells in lysis buffer composed of 20 mM Tris–HCl, pH 7.5, containing 150 mM NaCl, protease inhibitor cocktail (complete, Mini, EDTA-free, Roche), PhosSTOP phosphatase inhibitor cocktail (Roche) and Triton X-100 to a final concentration of 1%. Extracts were cleared from insoluble material by centrifugation for 10 min at 16,000×g, 4 °C.
For protein extraction from mouse tissues, hippocampi from 64 week-old mice (4–5 animals per group) were homogenized in 200 µl of 20 mM Tris–HCl, pH 7.5, containing 150 mM NaCl, protease inhibitor cocktail (complete, Mini, EDTA-free, Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche) using a dounce homogenizer as previously described^{3 (link)}. Proteins were solubilized by addition of Triton X-100 to a final concentration of 1%. Extracts were cleared from insoluble material by centrifugation for 30 min at 20,000×g, 4 °C. Protein concentration was determined with Quick Start Bradford 1 × Dye Reagent (BioRad Laboratories) as described by the manufacturer. n = 4–5 and three technical replicates were performed.

Ingwersen T., Linnenberg C., D’Acunto E., Temori S., Paolucci I., Wasilewski D., Mohammadi B., Kirchmair J., Glen R.C., Miranda E., Glatzel M, & Galliciotti G. (2021). G392E neuroserpin causing the dementia FENIB is secreted from cells but is not synaptotoxic. Scientific Reports, 11, 8766.

+ Open protocol

+ Expand

Phosphatase Treatment of GFP-Tagged Proteins

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

At ZT4, 6-day-old seedlings grown in LD in WL conditions were frozen and
the material was disrupted in a ball mill using metal beads. Plant material was
resuspended (50 mM Tris–HCl, 150 NaCl, 0.5% Triton X-100, 1×
cOmplete Protease Inhibitor Cocktail, 1× PhosSTOP phosphatase inhibitor
cocktail, Roche) and the supernatant was cleared by centrifugation and incubated
for 30 min with ChromoTek GFP-Trap. Beads were washed nine times (50 mM
Tris–HCl, 150 NaCl, 0.5% Triton X-100, 1× cOmplete Protease
Inhibitor Cocktail, 1× PhosSTOP phosphatase inhibitor cocktail, Roche)
and supernatant completely removed. Beads were washed once with 1×
NEBuffer for PMP and resuspended in the same buffer. λ-Phosphatase was
added and beads were incubated for 20 min at 30 °C. The reactions were
stopped by removal of supernatants and addition of 2× NuPAGE LDS Sample
Buffer (including 1.8% β-mercaptoethanol). For the respective negative
controls, λ-phosphatase was heat inactivated by boiling the enzyme for 10
min.

Willige B.C., Zander M., Yoo C.Y., Phan A., Garza R.M., Wanamaker S.A., He Y., Nery J.R., Chen H., Chen M., Ecker J.R, & Chory J. (2021). PHYTOCHROME-INTERACTING FACTORs trigger environmentally responsive chromatin dynamics in plants. Nature genetics, 53(7), 955-961.

+ Open protocol

+ Expand

Striatal Protein Expression and Phosphorylation

Cited 1 time

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

To determine STEP expression and phosphorylation levels, as well as p38 and ERK1/2 phosphorylation in 6-week and 1-year old striata, whole-cell lysates were prepared using a protocol similar to that described previously (Saavedra et al. 2011 (link)). The striatum was homogenized in lysis buffer (1% Triton X-100, 50mM Tris-HCl, 10mM EGTA, 150mM NaCl, pH 7.5) containing ‘complete’ protease and ‘phosSTOP’ phosphatase inhibitor cocktails (Roche diagnostics). Samples were subsequently centrifuged at 16,000g (20min, 4°C) and the supernatant stored at −80°C until use. This preparation contains solubilized cytosolic and membrane proteins but should exclude the postsynaptic density (PSD) fraction.
After treating slices with either TAT-myc, TAT-STEP, TAT-GluN2B9c or TAT-GluN2BAA peptides, whole-cell lysates were obtained using a protocol described previously (Fan et al. 2012 (link)). After treatment, slices were placed in ice-cold PBS with 5mM EDTA and 5mM EGTA and the striatal tissue dissected on ice. The tissue was subsequently homogenized in 1% NP40-containing lysis buffer (50mM Tris, 150mM NaCl, pH 7.4) containing ‘complete’ protease and ‘phosSTOP’ phosphatase inhibitor cocktails (Roche diagnostics). Striatal homogenates were solubilized using ultrasonication for 10s and rotated to lyse samples (> 2h, 4°C).

Gladding C.M., Fan J., Zhang L.Y., Wang L., Xu J., Li E.H., Lombroso P.J, & Raymond L.A. (2014). Alterations in Striatal-Enriched Protein Tyrosine Phosphatase (STEP) Expression, Activation and Downstream Signaling in Early and Late Stages of the YAC128 Huntington’s Disease Mouse Model. Journal of neurochemistry, 130(1), 145-159.

+ Open protocol

+ Expand

Detection of Phosphorylated Proteins

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

Monoclonal antibodies directed against GFP, HA, and Myc were from Santa Cruz Biotechnology (Heidelberg, Germany). Polyclonal antibodies directed against phospho-(Ser317) Chk1 and mouse monoclonal antibodies directed against phospho-(Thr180/Tyr182) p38 were from Cell Signaling Technology [Bionordika (Sweden) AB, Stockholm, Sweden]. Monoclonal antibodies directed against α-tubulin and phospho-(Tyr15) Cdc2 (Cdk1) were from Sigma-Aldrich (Sigma Aldrich AB). Monoclonal antibodies against Cdc2 were from Abcam (Cambridge, UK). For immunoblotting, protein extracts were prepared as previously described (Asp and Sunnerhagen, 2003 (link)) with the addition of 1× PhosStop phosphatase inhibitor cocktail (Roche Diagnostics Scandinavia AB, Bromma, Sweden). Proteins were separated by SDS-PAGE. Epitope-tagged proteins were detected with the appropriate monoclonal antibodies.

Alao J.P., Sjölander J.J., Baar J., Özbaki-Yagan N., Kakoschky B, & Sunnerhagen P. (2014). Caffeine stabilizes Cdc25 independently of Rad3 in S chizosaccharomyces pombe contributing to checkpoint override. Molecular Microbiology, 92(4), 777-796.

+ Open protocol

+ Expand

Western Blot Analysis of BDNF and FoxO1

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

Western blots were performed as described previously [43 ]. Briefly, the dissected mPFC samples were homogenized in a lysis buffer (Beyotime Biotechnology, Shanghai, China) with 1% phenylmethylsulfonyl fluoride (Sangon Biotech, Shanghai, China) and 1 × PhosSTOP phosphatase inhibitor cocktail (Roche Applied Science, Penzberg, Germany). The extracted proteins were separated on a 15% SDS-PAGE gel and transferred to PVDF membrane (Millipore, Massachusetts, USA). The membrane was blocked with 5% non-fat milk powder in TBST buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween20), followed by incubation with the following primary antibodies diluted in a blocking solution overnight at 4 °C: anti-BDNF (sc-546, 1:500, Santa Cruz Biotechnology, Dallas, TX, USA), anti-FoxO1 (1:500, #2880, Cell Signaling Technology, Danvers, MA, USA), or anti-β-actin antibody (1:1000, #4970, Cell Signaling Technology, Danvers, MA, USA). After washing, the membrane was incubated with IRDye 680LT donkey anti-rabbit IgG secondary antibodies (1:5000; 926–68,023, Li-COR Biosciences, Lincoln, NE, USA). The fluorescence was visualized and analyzed using an Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE, USA).

Liu J., Meng F., Dai J., Wu M., Wang W., Liu C., Zhao D., Wang H., Zhang J, & Li C. (2020). The BDNF-FoxO1 Axis in the medial prefrontal cortex modulates depressive-like behaviors induced by chronic unpredictable stress in postpartum female mice. Molecular Brain, 13, 91.

+ Open protocol

+ Expand

Immunoprecipitation and Western Blot Analysis

Cited 1 time

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

Immunoprecipitation methods were described previously [52 (link)]. MDA-MB-231 and HEK293T cell lysates were treated using RIPA lysis buffer supplemented with 1× inhibitor cocktail (Roche) and 1× Roche PhosSTOP phosphatase inhibitor cocktail (Roche). After centrifugation at 12,000 rpm for 15 min at 4 °C, cell debris was removed. Precleared lysates were incubated with indicated antibodies and normal IgG (as controls) overnight at 4 °C. 40 μl of protein A or protein G agarose (Santa Cruz Biotechnology, Inc.) were incubated with Lysates with rotation at 4 °C for 6 h. The complex was washed with RIPA buffer five times, resuspended with 60 μl 2× loading buffer, and cooked at 100 °C for 8 min, which was detected by western blot analysis.

Wang Z., Zhang L., Li B., Song J., Yu M., Zhang J., Chen C., Zhan J, & Zhang H. (2023). Kindlin-2 in myoepithelium controls luminal progenitor commitment to alveoli in mouse mammary gland. Cell Death & Disease, 14(10), 675.

+ Open protocol

+ Expand

Western Blot Analysis of Signaling Proteins

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

Cell lysates or tissues were treated using RIPA lysis buffer supplemented with 1× inhibitor cocktail (Roche) and 1× Roche PhosSTOP phosphatase inhibitor cocktail (Roche). Aliquots of 30 μg protein were separated by SDS-PAGE and transferred to the polyvinylidene fluoride (PVDF) membrane. Membranes were blocked in 5% non-fat powdered milk at room temperature for 1 h and incubated with specific antibodies overnight at 4 °C. Antibodies used for western blot analysis in this study were: rabbit anti-Kindlin-2 (Sigma, K3269, 1:1000 dilution); mouse anti-GAPDH (Zhong Shan Jin Qiao, TA-08, 1:1000 dilution); mouse anti-tubulin (Sigma, clone B-5-1-2, 1:1000 dilution); rabbit anti-DDDDK tag (Abcam, ab205606, 1:1000 dilution); rabbit anti-STAT3 (Abcam, ab68153, 1:1000 dilution); rabbit anti-pSTAT3-S727 (Abcam, ab32143, 1:1000 dilution); rabbit anti-pSTAT3-Y705 (Abcam, ab76315, 1:1000 dilution); rabbit anti-Dll1 (Abcam, ab10554, 1:1000 dilution); rabbit anti-JAK2 (Abcam, ab108596, 1:1000 dilution). After washing three times in TBST buffer, the membranes were incubated with appropriate HRP-conjugated secondary antibodies at room temperature for 1 h. The Super Signal Chemiluminescence kit (Thermo Fisher Scientific) was used to detect immobilized antibodies.

+ Open protocol

+ Expand

Mammary Gland Protein Extraction

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

FACS-sorted cells were directly lysed in RIPA buffer containing 1 × complete mini protease inhibitor cocktail (Roche) and 1 × Roche PhosSTOP phosphatase inhibitor cocktail (Roche). Whole mammary gland lysates were prepared by grinding tissue in liquid nitrogen and solubilizing in the RIPA buffer. The following primary antibodies were used for western blot analysis: anti-AURKA (mouse, clone 4/IAK1, BD; 1:500 dilution) and anti-beta-Actin (mouse, clone AC-15, Sigma; 1:5,000 dilution). All uncropped Western blots can be found in Supplementary Fig. 11.

Rios A.C., Fu N.Y., Jamieson P.R., Pal B., Whitehead L., Nicholas K.R., Lindeman G.J, & Visvader J.E. (2016). Essential role for a novel population of binucleated mammary epithelial cells in lactation. Nature Communications, 7, 11400.

+ Open protocol

+ Expand

Western Blot Analysis of Protein Targets

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

Procedures were performed as previously described.^{18 (link)} Briefly, after 24-h treatment of IMZ (1 μM) or vehicle control in basal medium supplemented with 10% FBS, cells were washed with ice-cold PBS and lysed with cell lysate buffer (Cell Signaling), containing 1 mM Pefabloc SC, cOmplete protease inhibitor cocktail, and PhosSTOP phosphatase inhibitor cocktail (Roche). Equal amounts of denatured proteins (40 μg) were separated by 8%–14% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes for immunoblotting using antibodies directed against the protein targets as described in Table S1. Blots were imaged with the Odyssey Fc imaging system (Li-Cor). Densitometric analysis was performed using Image Studio Lite software (Li-Cor).

Nguyen H., Koh J.Y., Li H., Islas‐Robles A., Meda Venkata S.P., Wang J, & Monks T.J. (2021). A novel imidazolinone metformin‐methylglyoxal metabolite promotes endothelial cell angiogenesis via the eNOS/HIF‐1α pathway. The FASEB Journal, 35(7), e21645.

+ 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!