Protease phosphatase inhibitor

Manufactured by Cell Signaling Technology

Sourced in United States, China

Protease/phosphatase inhibitor is a laboratory reagent used to inhibit the activity of proteases and phosphatases in biological samples. It helps preserve the integrity of proteins and phosphorylation states during sample preparation and analysis.

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Lab products found in correlation

Pvdf membrane, by Merck Group (7 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (7 mentions) Ripa buffer, by Thermo Fisher Scientific (7 mentions) Ripa buffer, by Cell Signaling Technology (4 mentions) Laemmli sample buffer, by Bio-Rad (4 mentions) Ripa buffer, by Merck Group (3 mentions) Bca assay, by Thermo Fisher Scientific (3 mentions) P akt, by Cell Signaling Technology (3 mentions) Gapdh, by Thermo Fisher Scientific (3 mentions) Bca protein assay kit, by Beyotime (3 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (3 mentions) Ripa lysis buffer, by Cell Signaling Technology (2 mentions) Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions) Cell lysis buffer, by Cell Signaling Technology (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Ecl reagent, by Merck Group (2 mentions) Cell fractionation kit, by Cell Signaling Technology (2 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) Chemiluminescent substrate kit, by Bio-Rad (2 mentions) Rapi buffer, by Solarbio (2 mentions)

Close spelling variants of this product

Protease/phosphatase inhibitor cocktail (304 citations) Protease inhibitors (97 citations) Protease and phosphatase inhibitors (94 citations) Protease and phosphatase inhibitor cocktail (63 citations) Phosphatase inhibitor (44 citations) Phosphatase and protease inhibitors (5 citations) Protease inhibitor and phosphatase inhibitor (5 citations) Protease and phosphatase inhibitors cocktail (4 citations) Protease/Phosphatase Inhibitor Cocktail (100X), (4 citations) Protease and phosphatase inhibitor cocktails I and II (3 citations)

53 protocols using protease phosphatase inhibitor

Protein Extraction and Western Blotting

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Lung tissue samples were lyophilized and weighed. Consistent amounts of each sample (10 mg) were digested in a fixed volume of cold radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) with protease/phosphatase inhibitors (Cell Signaling) for 1 hour at 4°C, followed by centrifugation at 14,000g for 30 min at 4°C to remove insoluble material. Cell pellets [SAECs and human umbilical cord endothelial cells (HUVECs)] were digested in RIPA buffer, and protein concentration was quantified via Bradford assay. Samples were reduced in Laemmli buffer (Novex, Life Technologies) for 10 min at 70°C. Samples were run on 4 to 12% polyacrylamide gels (Invitrogen); 30 μg of SAEC and HUVEC cell extracts were loaded as control. After electrophoresis, proteins were transferred to a nitrocellulose membrane or polyvinylidene difluoride membrane (Bio-Rad). Membranes were blocked for 1 hour in blocking/probing buffer (PBS with 5% nonfat dry milk, Bio-Rad; 0.1% Tween, Sigma-Aldrich). Primary antibodies were applied overnight in blocking/probing buffer, followed by 3× 10-min wash in PBS 0.1% Tween 20 (42 (link)). Secondary antibody was applied for 1 hour at room temperature at a dilution of 1:5000, followed by three washes, as described above. Protein signal was detected using an Odyssey 2.1 scanner. Antibody sources and dilutions are described in tables S1 and S2.

Dorrello N.V., Guenthart B.A., O’Neill J.D., Kim J., Cunningham K., Chen Y.W., Biscotti M., Swayne T., Wobma H.M., Huang S.X., Snoeck H.W., Bacchetta M, & Vunjak-Novakovic G. (2017). Functional vascularized lung grafts for lung bioengineering. Science Advances, 3(8), e1700521.

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Quantitative Western Blot Analysis of Cholesterol Regulators

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Protein lysates were collected at 4 °C in RIPA buffer (Sigma) supplemented with protease inhibitors (10uM leupeptin, 5ug/ml pepstatin A, 3ug/ml aprotinin, 25ug/ml ALLN, and 0.5mM PMSF). Conditioned growth medium was collected at 4 °C with protease/phosphatase inhibitors (Cell Signaling) and concentrated with Amicon Ultra-15 filters (30-kDa cut-off, EMD Millipore). Total protein concentration was determined by BCA method (ThermoFisher) and equal amounts of total protein were loaded onto 8% or 4–12% Bis-Tris Plus Gels (ThermoFisher). Following electrophoresis (100 V, 1h), proteins were transferred to iBlot® 2 Transfer Stacks, nitrocellulose membranes (ThermoFisher Scientific). Blots were probed overnight at 4 °C with 1:500 anti-HMG-CoA reductase (EMD Milipore, ABS229), 1:1,000 anti-APOE (Calbiochem, 178479), 1:200 anti-SREBP2 (Abcam, 30682), 1:1,000 anti-LAMP1 (Abcam, ab24170), or 1:700 anti-ABCA1 (Abcam, ab18180) followed by 1:2,000 HRP-conjugated secondary (Goat, life technologies, 611620; Rabbit, Vector Laboratories, PI-1000; Mouse, Vector Laboratories, PI-2000, 1 h at room temperature) and visualized with WesternBright™ ECL HRP Substrate reagents (Advansta) on the UVP System.

TCW J., Qian L., Pipalia N.H., Chao M.J., Liang S.A., Shi Y., Jain B.R., Bertelsen S.E., Kapoor M., Marcora E., Sikora E., Andrews E.J., Martini A.C., Karch C.M., Head E., Holtzman D.M., Zhang B., Wang M., Maxfield F.R., Poon W.W, & Goate A.M. (2022). Cholesterol and matrisome pathways dysregulated in astrocytes and microglia. Cell, 185(13), 2213-2233.e25.

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Western Blot Analysis of Liver Signaling

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Frozen livers (∼50 mg) were homogenized in or primary hepatocytes were directly lysed in ice-cold lysis buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 10% glycerol, 2% NP-40, 1 mmol/L EDTA, 20 mmol/L NaF, 30 mmol/L Na₄P₂O₇, 0.2% [w/v] SDS, and 0.5% [w/v] sodium deoxycholate) supplemented with protease/phosphatase inhibitors (Cell Signaling Technology). Protein concentration was assessed by bicinchoninic acid assay (Sigma-Aldrich). The following antibodies used in the study were all purchased from Cell Signaling Technology: FoxO1 C29H4, Akt, phosphorylated (p) Akt Thr308, glycogen synthase kinase 3β (GSK-3β), and pGSK-3β Ser9. Densitometric analysis was performed using ImageJ software (National Institutes of Health).

Cook J.R., Matsumoto M., Banks A.S., Kitamura T., Tsuchiya K, & Accili D. (2015). A Mutant Allele Encoding DNA Binding–Deficient FoxO1 Differentially Regulates Hepatic Glucose and Lipid Metabolism. Diabetes, 64(6), 1951-1965.

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EpCAM Detection in Adherent and Suspension Cells

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Adherent cells were removed from their flasks with trypsin or 20 mM EDTA in PBS, then washed several times in PBS before lysing in 1x Cell Lysis Buffer (Cell Signaling Technology; Danvers, MA) containing 1x protease/phosphatase inhibitors (Cell Signaling Technology) Suspension cells were similarly washed in PBS before lysis. Lysates were reduced with 10% 2-mercaptoethanol and run on 4–20% Tris-Glycine SDS-PAGE, transferred to PVDF membranes, then probed with a goat polyclonal anti-EpCAM antibody (R&D, Cat# AF960), followed by a donkey anti-goat 800CW antibody (Licor), after which the membranes were scanned on an Odyssey (Licor).
For cell surface biotinylation experiments, biotinylation was performed on cells after removal from the flask, using the Pierce Cell Surface Biotinylation and Isolation kit (ThermoFisher). Cells not incubated with biotin were processed in parallel as controls. After biotinylation, cells were lysed according to the manufacturer's instructions, and protein concentrations in lysates were quantified prior to purification of biotinylated proteins with NeutrAvidin agarose. The proportion of purified protein equivalent to the original mass of total lysate was run on SDS-PAGE, then probed with antibodies as described above.

Bagheri A., Culp P.A., DuBridge R.B, & Chen T.H. (2022). CRISPR/Cas9 disruption of EpCAM Exon 2 results in cell-surface expression of a truncated protein targeted by an EpCAM specific T cell engager. Biochemistry and Biophysics Reports, 29, 101205.

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Quantifying Phosphorylated Proteins in Skeletal Muscle

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Approximately 30 mg tibialis anterior muscle were lysed in RIPA buffer containing protease/phosphatase inhibitors (Cell Signaling Technology, #5872S) with TissueLyser. A total of 30 µg of protein were used for Western blotting. Primary antibodies against p-AKT (S473, Cell Signaling Technology, #4060, rabbit), AKT (Cell Signaling Technology, #4691, rabbit), PTEN (Cell Signaling Technology, #9188, rabbit), pp38 (Cell Signaling Technology, #4511S, rabbit), and p38 (Cell Signaling Technology, #8690S, rabbit) were used for Western blots. Quantification of Western blotting was performed using NIH Image J software. For unknown reasons, only in cases of skeletal muscle extracts, we were not able to detect clear signals for commonly used loading control markers such as β-actin upon reprobing the blots. Therefore, Ponceau S staining of the blots was performed to ensure relatively equal loading of proteins in each lanes. Because phosphorylated proteins are sometimes not equally recognized by antibodies that detect total proteins, we did not use total protein corresponding to phosphoprotein for normalization of phosphoproteins. Therefore, quantitative levels of phosphoprotein and total proteins of protein-of-interest are presented separately.

Wang R., Khatpe A.S., Kumar B., Mang H.E., Batic K., Adebayo A.K, & Nakshatri H. (2024). Mutant RAS-driven Secretome Causes Skeletal Muscle Defects in Breast Cancer. Cancer Research Communications, 4(5), 1282-1295.

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Isolation of Solubilized Mouse Brain Membranes

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Dissected mouse brain regions were homogenized in homogenization buffer (50 mM NaCl, 50 mM NaH₂PO₄, 2 mM EDTA, 2 mM EGTA, pH 7.4) on ice with 30 strokes of a Potter-Elvehjem glass homogenizer or disposable polypropylene pestles depending on the size of the region. Membrane fragments were isolated following centrifugation at 21,130 x g for 10 minutes at 4 °C. Membrane pellets were then homogenized in solubilization buffer (50 mM NaCl, 50 mM NaH₂PO₄, 2 mM EDTA, 2 mM EGTA, 2% Triton X-100, pH 7.4) with 40 strokes of a Potter-Elvehjem glass homogenizer or disposable polypropylene pestles and incubated for three hours at 4 °C with agitation to solubilize membrane-bound proteins. Following a second centrifugation at 21,130 x g for 10 minutes at 4 °C, the solubilized membrane fraction was recovered in the supernatant.
All buffers used to isolate the solubilized membrane fraction were supplemented with protease/phosphatase inhibitors (Cell Signaling Technology, cat. no. 5872). Protein content of solubilized membrane fractions was determined using a BCA assay (Thermo Scientific, cat. no. 23225).

Mulcahy M.J., Huard S.M., Paulo J.A., Wang J.H., McKinney S., Marks M.J., Henderson B.J, & Lester H.A. (2021). Protein profiling in the habenula after chronic (–)-menthol exposure in mice. Journal of neurochemistry, 158(6), 1345-1358.

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Evaluating 20(S)-Rh2 Effects on A549 Cells

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A549 cells were seeded in 10 cm discs at a density of 5 × 10⁶ cells per disc and incubated for 24 h; then, cells were incubated with different concentrations (15, 22, and 35 μM) of 20(S)-Rh2 for another 24 h. Cell samples were washed with the ice-cold PBS and lysed with RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) containing protease/phosphatase inhibitors (Cell Signaling Technology, Danvers, MA, USA). Cell protein lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). After being blocked with 5% skim milk or BSA dissolved in TBST buffer, the membranes were incubated with primary antibodies at 4°C overnight. Then, goat anti-rat IgG secondary antibody was added. Following the washing step of the TBST buffer, an ECL immunoblotting detection kit (Clinx Science Instrument Co., Ltd., Shanghai, China) was used to detect the visualization. Antibody against GAPDH was used as an internal control.

Liang Y., Zhao J., Zou H., Zhang J, & Zhang T. (2022). Identification of 20(S)-Ginsenoside Rh2 as a Potential EGFR Tyrosine Kinase Inhibitor. Oxidative Medicine and Cellular Longevity, 2022, 6119737.

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Isolation of Mouse Brain Membranes

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Dissected mouse brain regions were homogenized in ice cold hypotonic 0.1x KRH buffer (144 mM NaCl, 2.2 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, and 25 mM HEPES, pH 7.5) with 30 strokes of a Potter-Elvehjem glass homogenizer or disposable polypropylene pestles on ice. Membrane fragments were isolated following centrifugation at 21,130 x g for 10 minutes at 4 °C. Pellets were washed three more times with 0.1x KRH to remove substances that could compete with epibatidine binding.
All buffers were supplemented with protease/phosphatase inhibitors (Cell Signaling Technology, cat. no. 5872). Protein content of the washed homogenates were determined using a BCA assay (Thermo Scientific, cat. no. 23225).

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Protein Extraction and Western Blot

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Cells were seeded in 12-well plates at 10⁶ cells/ml in 1 ml in culture medium and drugs were added for various period of time. Cells were recovered, spun down for 5 min at 500×g and pellets were resuspended in 1x RIPA buffer (Bio Basic Inc., Markham, Canada)/cat. no. RB4478) containing protease/phosphatase inhibitors (Cell Signaling Technologies/cat. no. 5872S) and kept on ice for lysis for 20 min. Cleared cell lysates were obtained after 5-min centrifugation at 14 000×g. Cell lysates were dosed for protein concentration using a BCA protein assay kit (Thermo Scientific, Zug, Switzerland)/cat. no. 23225). Thirty microgram of protein was loaded on 8% or 12% SDS-PAGE. Standard western blot procedure was followed.

Martin R., Desponds C., Eren R.O., Quadroni M., Thome M, & Fasel N. (2016). Caspase-mediated cleavage of raptor participates in the inactivation of mTORC1 during cell death. Cell Death Discovery, 2, 16024-.

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Protein Expression Analysis by Western Blot

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Methods have been described previously.15 (link)–17 (link) In brief, after each treatment, whole-cell lysates were prepared by sonication in Cellytic MT buffer (Sigma-Aldrich) with protease/phosphatase inhibitors (Cell Signaling Technology) and cleared by centrifugation. Samples consisting of 40 µg proteins were resolved on a denaturing 4%–20% SDS-PAGE gel (Bio-Rad) and transferred to polyvinylidene fluoride membranes by electroblotting. Membranes were then blocked in PBS blocking buffer (Li-Cor) for 1 hour and incubated with specific primary antibodies at 4°C overnight. Blots were then incubated with species-specific secondary antibodies (Cell Signaling Technology). Signals were detected by a chemiluminescence reagent and analyzed with ImageJ software.

Gong T., Yu Y., Yang B., Lin M., Huang J.W., Cheng B, & Ji C. (2018). Celecoxib suppresses cutaneous squamous-cell carcinoma cell migration via inhibition of SDF1-induced endocytosis of CXCR4. OncoTargets and therapy, 11, 8063-8071.

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