Proteases and phosphatases inhibitors

Manufactured by Merck Group

Sourced in United States

Proteases and phosphatases inhibitors are a class of laboratory equipment used to control the activity of enzymes that cleave proteins (proteases) or remove phosphate groups (phosphatases) from other molecules. These inhibitors are commonly used in various experimental settings to regulate the function of these enzymes and study their roles in biological processes.

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Lab products found in correlation

Polyvinylidene difluoride membrane, by Merck Group (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Turbofect, by Thermo Fisher Scientific (2 mentions) Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions) Restoretm plus western blot stripping buffer, by Thermo Fisher Scientific (1 mentions) Chemidoc mp system, by Bio-Rad (1 mentions) Dry skim milk, by Bio-Rad (1 mentions) Clarity enhanced chemiluminescence kit, by Bio-Rad (1 mentions) Ripa lysis buffer, by Cell Signaling Technology (1 mentions) Image lab software, by Bio-Rad (1 mentions) Magnesphere, by Promega (1 mentions) Epha3 c 19, by Santa Cruz Biotechnology (1 mentions) β actin, by Merck Group (1 mentions) Mission microrna mimic, by Merck Group (1 mentions) M per extraction buffer, by Thermo Fisher Scientific (1 mentions) Proteases inhibitors, by Merck Group (1 mentions) Bromophenol blue, by Merck Group (1 mentions) Laemmli buffer, by Merck Group (1 mentions) Ultrasonic, by Emerson (1 mentions)

8 protocols using proteases and phosphatases inhibitors

Cell Lysis and Protein Extraction Protocol

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Cells were washed two times with pre-chilled phosphate-buffered saline (PBS) purchased from Sigma-Aldrich and were lysed.
In the case of MEFs, a commercial lysis Buffer 10× (Cell Signaling Technology, Danvers, MA, USA) complemented with 2% sodium dodecyl sulfate (SDS) and proteases’ inhibitors (Sigma-Aldrich) was used. Cells were also sonicated (Branson Ultrasonic, Carouge, Switzerland) for 10 s at 50% amplitude.
In the case of HEK 293 cells, two different receipts were used as follows: (i) for the Beclin-1-c-FLIP co-immunoprecipitation: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM dithiothreitol, 0.5% Triton, 1 mM EDTA pH 8, proteases and phosphatases inhibitors, all purchased from Sigma-Aldrich; (ii) for the ubiquitination and the competition assay: 50 mM Tris-HCl pH 7.5, 1% Triton, 0.25% Na-Deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA pH 8, 5 mM MgCl₂, proteases and phosphatases inhibitors, all purchased from Sigma-Aldrich. Cells were then incubated for 30 min on ice.
Lysates from both cell lines were then centrifuged at 4 °C for 10 min at 13,000 × g to remove cell debris.
Protein concentration was determined by micro BCA assay (Pierce, Rockford, IL, USA) and samples were boiled at 95 °C for 10 min following Laemmli Buffer addition (0.04% Bromophenol blue, 40% Glycerol, 2% SDS, 20% β-mercaptoethanol, 250 mM Tris-HCl pH.6.8, all purchased from Sigma-Aldrich.

Tomaipitinca L., Petrungaro S., D’Acunzo P., Facchiano A., Dubey A., Rizza S., Giulitti F., Gaudio E., Filippini A., Ziparo E., Cecconi F, & Giampietri C. (2021). c-FLIP regulates autophagy by interacting with Beclin-1 and influencing its stability. Cell Death & Disease, 12(7), 686.

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Western Blot Protein Analysis

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Proteins were extracted in RIPA buffer supplemented with a cocktail of proteases and phosphatases inhibitors (all from Sigma) and concentration was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific). Primary antibodies (online supplementary Table S3) were incubated for 16 h at 4°C. Either β‐actin or β‐tubulin were used as loading control. Anti‐rabbit or ‐mouse IgG HRP‐conjugated were employed as secondary antibodies (both 1:5000, GE Healthcare). Membrane development was performed by an enhanced chemiluminescence‐based detection method (ECL™ Prime Western Blotting Detection Reagent, GE Healthcare) in a ChemiDoc‐MP system (Bio‐Rad). No more than one stripping procedure was performed on an individual membrane (RestoreTM Plus Western Blot Stripping Buffer, Thermo Fisher Scientific).

Dang Z., Avolio E., Thomas A.C., Faulkner A., Beltrami A.P., Cervellin C., Carrizzo A., Maciag A., Gu Y., Ciaglia E., Finato N., Damato A., Spinetti G., Alenzi A., Paisey S.J., Vecchione C., Puca A.A, & Madeddu P. (2020). Transfer of a human gene variant associated with exceptional longevity improves cardiac function in obese type 2 diabetic mice through induction of the SDF‐1/CXCR4 signalling pathway. European Journal of Heart Failure, 22(9), 1568-1581.

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Western Blot Protein Analysis Protocol

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Cultured cells were washed twice before lysis with ice-cold 1 × PBS (phosphate-buffered saline). Cells were then lysed using RIPA (radioimmunoprecipitation assay) buffer supplemented with proteases and phosphatases inhibitors (Sigma-Aldrich, Sigma, St Louis, MO, USA). Protein concentration in lysates was determined by a Pierce BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Equal amount of protein between samples was separated by electrophorese on SDS–PAGE gel and transferred to polyvinylidene difluoride membranes (Merck Millipore, USA). Membranes were incubated with 5% non-fat milk in TBS (Tris-buffered saline) buffer for an hour at room temperature and then incubated with primary antibodies overnight at 4 °C. The membranes were washed three times with 1 × TBS buffer before incubation with LICOR 680 nm or 800 nm fluorescent secondary antibodies for one hour. After washing with 1 × TBS buffer, the membranes were scanned on a LICOR Odyssey system. The acquired images were analyzed with Image Studio Version 4.0 software according to manufacturer’s instructions.

Zhang Z., Deng Y., Zheng G., Jia X., Xiong Y., Luo K., Qiu Q., Qiu N., Yin J., Lu M., Liu H., Gu Y, & He Z. (2017). SRGN-TGFβ2 regulatory loop confers invasion and metastasis in triple-negative breast cancer. Oncogenesis, 6(7), e360-.

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Western Blot Analysis of Autophagy Markers

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Mice kidneys were homogenized and incubated with radio-immunoprecipitation assay (RIPA) lysis buffer (Cell Signaling, USA) containing proteases and phosphatases inhibitors (Sigma, Israel) in ratio of 1 : 100 and 1 : 1000, respectively. Kidney's lysate was centrifuged, and the supernatant containing total proteins was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. Membranes were then blocked with 5% dry skim milk (Bio-Rad, CA, USA) in Tris-buffered saline with Tween 20 at room temperature and washed and incubated with primary antibodies (Anti-APG5L/ATG5 ab109490 [EPR4797] 1 : 1000; Anti-LC3-II ab48394 1 : 1000; Anti-GAPDH ab181602 1 : 10000) at 4°C overnight. Membranes were incubated with horseradish peroxidase-conjugated to secondary antibody at room temperature. Bands were visualized using clarity enhanced chemiluminescence kit (Bio-Rad, CA, USA) and were analyzed using Bio-Rad image Lab software, are data presented in arbitrary units (AU).

Saad R., Tadmor H., Ertracht O., Nakhoul N., Nakhoul F., Evgeny F, & Atar S. (2022). The Molecular Effects of SGLT2i Empagliflozin on the Autophagy Pathway in Diabetes Mellitus Type 2 and Its Complications. Journal of Diabetes Research, 2022, 8337823.

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Magnetic Bead Capture of Protein-RNA Complexes

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Streptavidin-conjugated paramagnetic beads (Z5481, Magnesphere, Promega) were washed three times in PBS according to the manufacturer's instructions, and then mixed with 450 μg of A375 EV proteins, in 500 μl (final volume) of binding buffer (BB: 75 mM Tris-HCl, pH 7.5; 50 mM KCl; 5 mM dithiothreitol) (27 (link)), containing proteases and phosphatases inhibitors (Sigma-Aldrich). Samples were incubated for 1 h, at 4°C, under shaking, to allow unspecific protein binding to the particles (pre-clearing step). After centrifuging at 10,000 × g for 5 min, the pre-cleared supernatants were used for the specific binding reaction. The pre-cleared sample was divided into two aliquots, one of which was mixed with H1.0 RNA (1.2 μg) in BB, while the other one was used as an RNA-free control. Both samples were incubated for 1 h at 4°C, after which fresh aliquots of pre-washed beads were added, and incubation was continued for 1 h, at 4°C, under shaking. Finally, the supernatants containing unbound proteins were collected by a magnetic device (Magnesphere, Promega) and frozen. Paramagnetic beads were washed four times in BB and then resuspended in electrophoresis sample buffer, boiled and centrifuged at 10,000 × g. The supernatants, which contain bound proteins, were frozen and saved for analyses.

Schiera G., Di Liegro C.M., Puleo V., Colletta O., Fricano A., Cancemi P., Di Cara G, & Di Liegro I. (2016). Extracellular vesicles shed by melanoma cells contain a modified form of H1.0 linker histone and H1.0 mRNA-binding proteins. International Journal of Oncology, 49(5), 1807-1814.

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Western Blot of Eph/ephrin Proteins

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Cell lysates were prepared by lysing cells in RIPA buffer with proteases and phosphatases inhibitors (Sigma), and separated by 10% SDS-PAGE. Western blotting was performed as previously described [15 (link)]. Primary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) included: EphA3 (C-19), EphA3 (L-18), ephrin-A5 (RR-7), and ephrinA1 (V-18). Other antibodies used were EphA2 (clone D7) (EMD Millipore Corporation, Billerica, MA) and β-actin (Sigma).

Ferluga S., Tomé C.M., Herpai D.M., D'Agostino R, & Debinski W. (2016). Simultaneous targeting of Eph receptors in glioblastoma. Oncotarget, 7(37), 59860-59876.

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MicroRNA-181a Functional Assay

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Cells were seeded at ~70% confluency in 6-well plates and, 24 h later, transfected with 100nM hsa-miR-181a or human negative control 2 (MISSION microRNA Mimic, Sigma-Aldrich) using turbofect according to manufacturer’s instructions (Thermo Scientific). 18 h later, media was replaced. After 48 h of total incubation time, cells were washed once with PBS, lysed with M-Per extraction buffer (200uL, Thermo Fisher Scientific) complemented with proteases and phosphatases inhibitors (Sigma-Aldrich), incubated 5 min at RT with gently agitation and centrifuged at 14,000 RPM for 10 min at 4°C. Immunoblot or RT-qPCR was performed as described above.

Rodriguez-Ortiz C.J., Baglietto-Vargas D., Martinez-Coria H., LaFerla F.M, & Kitazawa M. (2014). Up-regulation of miR-181 decreases c-Fos and SIRT-1 in the hippocampus of 3xTg-AD mice. Journal of Alzheimer's disease : JAD, 42(4), 1229-1238.

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Protein Extraction and Western Blot Analysis

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Cells were harvested, washed and the total proteins were extracted in ice-cold RIPA buffer supplemented with proteases and phosphatases inhibitors (Sigma-Aldrich, Sigma, USA). The protein concentration was quantified using the Pierce Protein Assay Kit (Pierce, 23227). For western blot assay, an equal amount of protein was separated by electrophoresis on SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Merck Millipore, USA). Membranes were incubated with 5% non-fat milk in TBS (Tris-buffered saline) buffer and probed with various primary antibodies HIF1α (Abcam, ab113642, 1:1000); SRGN (Abcam, ab211515, 1:500); β-actin, GAPDH, β-Tubulin (Beijing Ray Antibody Biotech, RM2003, RM2002, RM2001, 1:5000). The membranes were washed three times with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, anti-rabbit IgG 7074S or antimouse IgG 7076S, 1:5000). Specific antibody binding was detected using enhanced chemiluminescence detection reagents (Pierce).

Xu Y., Xu J., Yang Y., Zhu L., Li X, & Zhao W. (2018). SRGN Promotes Colorectal Cancer Metastasis as a Critical Downstream Target of HIF-1α. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 48(6).

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