Phosstop and complete phosphatase protease inhibitor cocktails

Manufactured by Roche

Sourced in United States

PhosSTOP and Complete Phosphatase/Protease Inhibitor Cocktails are laboratory reagents designed to inhibit the activity of phosphatases and proteases. They are used to preserve the phosphorylation state and prevent protein degradation in biological samples during extraction and analysis.

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5 protocols using phosstop and complete phosphatase protease inhibitor cocktails

Western Blot Analysis of Cell Signaling Pathways

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Total protein
extracts were generated using lysis buffer (50 mM Tris-HCl, pH 7.4,
150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1
mM EDTA) supplemented with PhosSTOP and Complete Phosphatase/Protease
Inhibitor Cocktails (Roche Diagnostics GmbH, Mannheim, Germany). Protein
extracts (20–25 μg per sample) were loaded onto sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels
and transferred electrophoretically to poly(vinylidene fluoride) (PVDF)
membranes. The following primary antibodies were used at a dilution
of 1:1000: ERK1/2 (p44/42) (ab17942, Abcam), p-ERK1/2 (p-p44/42) (Thr202/Tyr204)
(9101, Cell Signaling), MNK1 (2195, Cell Signaling), p-MNK1 (Thr197/202)
(2111, Cell Signaling), eIF4E (9742, Cell Signaling), p-eIF4E (p-Ser
209) (9741, Cell Signaling or NBP2-66802, Novus Biologicals), p38alpha
(9218, Cell Signaling), p38beta (2339, Cell Signaling), p-p38 (4511,
Cell Signaling), p-ATF2 (27934, Cell Signaling), Hsp27 (2402, Cell
Signaling), p-Hsp27 (S82) (2401, Cell Signaling), and p-p90 RSK (Thr573)
(9346, Cell Signaling). The primary HRP-conjugated antibody anti-β-actin
(Calbiochem) was used at a dilution of 1:20 000. Anti-mouse
and anti-rabbit HRP secondary antibodies were from Pierce and used
at a dilution of 1:10 000. Immunodetection of proteins was
performed using ECL Western Blotting Detection Reagents (GE Healthcare,
Buckinghamshire, U.K.).

Bou-Petit E., Hümmer S., Alarcon H., Slobodnyuk K., Cano-Galietero M., Fuentes P., Guijarro P.J., Muñoz M.J., Suarez-Cabrera L., Santamaria A., Estrada-Tejedor R., Borrell J.I, & Ramón y Cajal S. (2022). Overcoming Paradoxical Kinase Priming by a Novel MNK1 Inhibitor. Journal of Medicinal Chemistry, 65(8), 6070-6087.

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Protein Extraction and Western Blotting Procedure

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Total protein extracts were generated using RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) supplemented with PhosSTOP and Complete Phosphatase/Protease Inhibitor Cocktails (Roche Diagnostics GmbH, Mannheim, Germany). Protein extracts (20–25 μg per sample) were loaded onto gels and immunodetection of proteins was performed using ECL™ Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire UK).

Castellana B., Aasen T., Moreno-Bueno G., Dunn S.E, & Ramón y Cajal S. (2015). Interplay between YB-1 and IL-6 promotes the metastatic phenotype in breast cancer cells. Oncotarget, 6(35), 38239-38256.

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Western Blot Protein Analysis

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Total protein extracts were generated using lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) supplemented with PhosSTOP and Complete Phosphatase/Protease Inhibitor Cocktails (Roche Diagnostics GmbH, Mannheim, Germany). Protein extracts (20–25 μg per sample) were loaded onto SDS-PAGE gels and transferred electrophoretically to PVDF membranes and immunodetection of proteins was performed using ECL™ Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK). The following primary antibodies were used: anti-Myc, anti-HIF1a, anti-4EBP1, anti-eIF4E, anti-YBX1, anti-Snail, anti-CA9, anti-Smad2/3, anti-β-catenin (Cell Signaling), anti-phospho Smad2 (Millipore), anti-β3 integrin, anti-MMP3, anti-N-cadherin (Abcam), anti-CycD1, anti-vimentin (Santa Cruz Biotechnology), anti-BMP2, anti-CCDC103, anti-TTC30B, anti-EIF3G, anti-RPL11 (CusaBio), anti-Cx31 (Alpha Diagnostics), anti-eIF4E2 (GeneTex), anti-αv integrin and anti-β-actin (1:500; Calbiochem, Darmstadt, Germany). Anti-mouse and anti-rabbit HRP secondary antibodies were from Pierce. Bound antibodies were visualized with an enhanced chemiluminescence detection kit (Amersham Pharma-Biotech).

Sesé M., Fuentes P., Esteve-Codina A., Béjar E., McGrail K., Thomas G., Aasen T, & Ramón y Cajal S. (2017). Hypoxia-mediated translational activation of ITGB3 in breast cancer cells enhances TGF-β signaling and malignant features in vitro and in vivo. Oncotarget, 8(70), 114856-114876.

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Cell Lysis and Protein Quantification

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Cells were lysed with radioimmunoprecipitation assay (RIPA) Lysis Buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM ethylenediaminetetraacetic acid (EDTA)) (Santa Cruz Biotechnology, Dallas, TX, USA) supplemented with PhosSTOP and Complete Phosphatase/Protease Inhibitor Cocktails (Roche Diagnostics GmbH, Mannheim, Germany). Protein was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).

Tishchenko A., Azorín D.D., Vidal-Brime L., Muñoz M.J., Arenas P.J., Pearce C., Girao H., Ramón y Cajal S, & Aasen T. (2020). Cx43 and Associated Cell Signaling Pathways Regulate Tunneling Nanotubes in Breast Cancer Cells. Cancers, 12(10), 2798.

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Western Blot Analysis of Neuronal Markers

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Western blot analysis was performed as previously reported (Jiang et al., 2016 (link); Bier et al., 2018 (link)). Briefly, cell lysates were solubilized with RIPA buffer supplemented with PhosSTOP and Complete Phosphatase/Protease Inhibitor Cocktails (Roche Diagnostics) and protein content was analyzed using a standard BCA assay. Protein extract (20–30 μg per sample) were loaded on sodium dodecyl sulfate-polyacrylamide electrophoresis gels and transferred to PVDF membranes that were probed with the specific antibodies as detailed. Bound antibodies were visualized with an enhanced chemiluminescence detection kit (Amersham Pharma-Biotech). Equal loading was verified using an anti actin antibody. The following primary antibodies were used: Anti-TrkB (Cat# sc-136990, 1:500), anti-p75 (Cat# sc-13577, 1:1,000), and anti-EAAT2 antibodies (Cat# sc-365634, 1:500) (Santa Cruz Biotechnology). Anti-cleaved PARP1 (AB3820, 1:1,000), Anti-cleaved caspase 3 (ab2303, 1:500), anti-C3 (ab97462, 1:500) anti-S100A10 (ab76472, 1:500) and exosome panel antibody (ab275018) (Abcam, Cambridge, MA United States). Anti-mouse and anti-rabbit HRP secondary antibodies (1:10,000, Pierce).

Penning D.H., Cazacu S., Brodie A., Jevtovic-Todorovic V., Kalkanis S.N., Lewis M, & Brodie C. (2021). Neuron-Glia Crosstalk Plays a Major Role in the Neurotoxic Effects of Ketamine via Extracellular Vesicles. Frontiers in Cell and Developmental Biology, 9, 691648.

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