Protease and phosphatase inhibitors cocktail

Manufactured by Merck Group

Sourced in United States, China, France

Protease and phosphatase inhibitors cocktail is a laboratory reagent designed to inhibit the activity of proteases and phosphatases in biological samples. It is used to prevent the degradation of proteins and to maintain the phosphorylation state of proteins during sample preparation and analysis.

Automatically generated - may contain errors

Lab products found in correlation

Clarity western ecl substrate, by Bio-Rad (4 mentions) Pvdf membrane, by Merck Group (3 mentions) Ripa buffer, by Beyotime (3 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions) Anti akt, by Cell Signaling Technology (2 mentions) Image lab software, by Bio-Rad (2 mentions) Psma7, by Thermo Fisher Scientific (2 mentions) Actin hrp, by Merck Group (2 mentions) Uchl1, by R&D Systems (2 mentions) Uchl1, by Cell Signaling Technology (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Immobilon p pvdf membrane, by Merck Group (2 mentions) T per, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Np40 lysis buffer, by Thermo Fisher Scientific (1 mentions) Mini trans blot cell, by Bio-Rad (1 mentions) P150glued, by BD (1 mentions) Anti phospho gsk3β ser9, by Cell Signaling Technology (1 mentions) Anti phospho akt thr308, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

Protease inhibitor cocktail (9861 citations) Protease inhibitors (2755 citations) Phosphatase inhibitor cocktail (1110 citations) Phosphatase inhibitors (852 citations) Protease inhibitors cocktail (264 citations) Protease/phosphatase inhibitors (43 citations) Proteases inhibitor cocktail (23 citations) Phosphatase inhibitors cocktail (20 citations) Proteases Inhibitors Cocktail (8 citations) Cocktail inhibitor (6 citations)

48 protocols using protease and phosphatase inhibitors cocktail

Chromatin-Bound Protein Extraction Protocol

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

The protein extraction protocol used to detect the chromatin bound fraction of relevant proteins involved in the NER process has been previously described (24) . Briefly, the pellets obtained as described above were resuspended in hypotonic lysis buffer (10 mM Tris-HCl at pH 8.0, 2.5 mM MgCl 2 , 0.5% Igepal, 0.2 mM Na 3 VO 4 , 1 mM dithiothreitol (DTT), 10 mM β-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), protease and phosphatase inhibitors cocktails (Sigma).
After washing once in hypotonic buffer, and then in isotonic buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM PMSF, 1 mM DTT, 10 mM β-glycerophosphate, protease and phosphatase inhibitors cocktails), the pellet was re-suspended in DNase I digestion buffer (10 mM Tris-HCl pH 8.0, 5 mM MgCl 2 , 10 mM NaCl, 1 mM PMSF, protease inhibitor cocktail) and incubated for 20 min at 4°C. Released proteins were mixed in 3X loading buffer, boiled at 75 °C and stored at -20°C.
Proteins were separated on NuPAGE 4-12% Bis-Tris gels, and transferred to nitrocellulose membranes for subsequent immunoblot analysis with relevant primary antibodies, and HRPconjugated secondary antibodies. Nitrocellulose membranes were incubated with substrate Clarity (Bio-Rad) and chemiluminescence detection was performed with a Westar R Chemiluminescence Imager (HiTech Cyanagen) for digital acquisition of images.

Scalera C., Dutto I., Barbazza F., Khouzam R.A., Ticli G., Cazzalini O., Stivala L.A., & Prosperi E. (2020). Dynamics of protein binding to sites of nascent unscheduled DNA repair synthesis in non-proliferating cells.

+ Open protocol

+ Expand

Protein Partitioning in C2C12 Myotubes

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

To estimate protein partitioning between Triton-soluble (cytosolic) and Triton-insoluble (myofibrillar) fractions of C2C12 cells, a six-well of cultures of differentiated myotubes was washed twice in ice-cold PBS (1 mL) and then scraped in 500 μL. Cells were centrifuged at 12000×g for 10 min at +4 °C and the pellet was lysed in 100 μL detergent-soluble fraction (DSF) buffer containing 10 mM Tris-HCl (pH 7.5), 1% Triton X-100, 5 mM EDTA and supplemented with protease and phosphatase inhibitors cocktail (Sigma). Insoluble material was recovered by a centrifugation at 16000g for 15′ at 4 °C. The supernatant (soluble fraction) was collected in new tubes, while the pellet, was then washed with supplemented DSF buffer and centrifuged at 16000 g for 15′ at 4 °C. The insoluble materials represented by pellets were resuspended in 50 μl detergent-insoluble fraction (DIF) buffer enclosing 10 mM Tris-HCl (pH 7.5), 1% SDS and supplemented with protease and phosphatase inhibitors cocktail (Sigma), incubated for 15 min at room temperature and for 2 min on ice, and sonicated once at 50% amplitude for 10 sec on ice (Vibra-Cell, Sonics & Materials). Equal amounts of each fraction were heated for 5 min at 95 °C in 4 × Laemmli Sample Buffer and analysed by SDS-PAGE.

Dimauro I., Antonioni A., Mercatelli N., Grazioli E., Fantini C., Barone R., Macaluso F., Di Felice V, & Caporossi D. (2019). The early response of αB-crystallin to a single bout of aerobic exercise in mouse skeletal muscles depends upon fiber oxidative features. Redox Biology, 24, 101183.

+ Open protocol

+ Expand

Western Blot for protein detection

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

Cells were harvested with NP-40 lysis buffer (ThermoFisher Scientific, UK) with protease and phosphatase inhibitors cocktails (Sigma‐Aldrich). Samples were loaded on 7.5% or 4–20% gels and resolved by electrophoresis. Proteins were transferred to PVDF membranes using Mini Trans-Blot Cell (BioRad). Membranes incubated for 1 h in blocking solution consisting of 5% w/v milk diluted in TBS-T 145 mM NaCl, 20 mM Tris-base, pH 7.4, 0.5% Tween-20 and labelled with primary antibody overnight at 4 °C for DHC (Proteintech—1:1000), Rab46 (CRACR2A: Proteintech 1:800), p150^Glued (BD Biosciences—1:1000), ATP1α1 (Santa Cruz—1:500), GFP (ThermoFisher—1:1000) and histidine (BioRad—1:1000). Immunoblots visualised using HRP-conjugated donkey anti-mouse, anti-rabbit secondary antibodies (Jackson ImmunoResearch—1:10,000) and SuperSignal Femto (Pierce).

Pedicini L., Wiktor S.D., Simmons K.J., Money A, & McKeown L. (2021). Affinity-based proteomics reveals novel binding partners for Rab46 in endothelial cells. Scientific Reports, 11, 4054.

+ Open protocol

+ Expand

Western Blot Analysis of Cell Signaling Pathways

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

The following antibodies were used: anti-GAPDH (1:5000) and anti-CDKL5 (1:500) (Sigma); anti-phosphorylated Erk1/2 (1:1000), anti Erk1/2 (1:1000), anti-phospho-AKT-Ser⁴⁷³ (1:1000), anti-phospho-AKT-Thr³⁰⁸ (1:1000), anti-AKT (1:1000), anti-phospho-GSK-3β-Ser⁹ (1:1000), anti-GSK-3β (1:1000), anti-phospho-CRMP2-Thr⁵¹⁴ (1:1000) and anti-CRMP2 (1:1000) (Cell Signaling Technology); anti β-catenin (1:1000; BD Transduction Laboratories); anti-phospho-CREB-Ser¹³³ (1:1000) and anti-CREB (1:1000) (Upstate Biotechnology). For the preparation of total cell extracts, cells were lysed in RIPA buffer (Tris–HCl 50 mM, NaCl 150 mM, Triton X-100 1%, sodium deoxycholate 0.5%, SDS 0.1%, protease and phosphatase inhibitors cocktails 1%; Sigma). For the preparation of tissue extracts, the hippocampi from P19 mice were homogenized in RIPA buffer. Extracts were immediately processed by Western blot or kept frozen (− 80 °C) until assayed. Sample protein concentration was estimated using the Lowry method (Lowry et al., 1951 (link)). Equivalent amounts (50 μg) of protein were subjected to electrophoresis on a 10% SDS-polyacrylamide gel. Densitometric analysis of digitized images was performed using Scion Image software (Scion Corporation, Frederick, MD, USA) and intensity for each band was normalized to the intensity of the respective total protein levels or GAPDH band.

Fuchs C., Trazzi S., Torricella R., Viggiano R., De Franceschi M., Amendola E., Gross C., Calzà L., Bartesaghi R, & Ciani E. (2014). Loss of CDKL5 impairs survival and dendritic growth of newborn neurons by altering AKT/GSK-3β signaling. Neurobiology of Disease, 70(100), 53-68.

+ Open protocol

+ Expand

Western Blot Analysis of Cell Signaling

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

Protein extraction was performed using a RIPA Lysis buffer supplemented with protease and phosphatase inhibitors cocktails (Sigma-Aldrich, P0044) while protein concentration was determined using the Bradford assay (Bio-Rad, 5000006). Briefly, western blot samples were exposed to denaturation then migrated through 8, 12 and 15% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and 5% milk solution was used for blocking purposes. The primary antibodies were added overnight after being diluted in 1% milk solution: p53 (Santa cruz, sc263, 1:200), p21 (Santa cruz, sc6246, 1:100), p27 (Santa cruz, sc71813, 1:100), cyclin D1 (Santa cruz, sc8396, 1:200), Noxa (Santa cruz, sc515840, 1:100), PARP1 (Santa cruz, sc8007, 1:200), GAPDH (Santa cruz, sc47724, 1:1000). Membranes were then incubated with the goat anti-mouse HRP conjugate secondary antibody (Bio-Rad, 1706515, 1:2000) prepared in 1% milk solution and this for 1 h before being exposed to the Clarity western ECL substrate (Bio-Rad, 1705061). Band detection was enabled using the Bio-Rad VersaDoc Imaging system and quantification was done by the means of the ImageJ software. Three to four biological replicates were carried out.

Issa H., Loubaki L., Al Amri A., Zibara K., Almutairi M.H., Rouabhia M, & Semlali A. (2024). Eugenol as a potential adjuvant therapy for gingival squamous cell carcinoma. Scientific Reports, 14, 10958.

+ Open protocol

+ Expand

Cell Viability and Apoptosis Assays

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

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), crystal violet, propidium iodide (PI), RNAse, cell lysis buffer, dimethyl sulfoxide (DMSO), phenylmethylsulfonyl fluoride (PMSF), protease and phosphatase inhibitors cocktails were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse monoclonal anti-PARP antibody was acquired from Cell Signaling Technology, Inc. (Beverly, MA, USA). Monoclonal anti-β-actin antibody was acquired from Sigma-Aldrich (St. Louis, MO, USA). Anti-phospho-histone H2A.X (Ser139), anti-cyclin B1, goat anti-mouse IgG-horseradish peroxidase (HRP) and goat anti-rabbit IgG-HRP antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Florio R., Veschi S., di Giacomo V., Pagotto S., Carradori S., Verginelli F., Cirilli R., Casulli A., Grassadonia A., Tinari N., Cataldi A., Amoroso R., Cama A, & De Lellis L. (2019). The Benzimidazole-Based Anthelmintic Parbendazole: A Repurposed Drug Candidate That Synergizes with Gemcitabine in Pancreatic Cancer. Cancers, 11(12), 2042.

+ Open protocol

+ Expand

Western Blot Analysis of Retinal Proteins

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

Mice were sacrificed by CO₂ inhalation and retinas dissected and lysed in RIPA Lysis Buffer with protease and phosphatase inhibitors cocktails (Sigma-Aldrich). Protein samples (20 μg) were run on 10% (w/v) SDS-PAGE gel and then transferred to PVDF membranes (Millipore, Watford, UK). Samples were then probed for primary antibodies (including β-actin loading controls; Supplemental Table 1) followed by incubation with appropriate HRP-conjugated secondary antibodies, and imaged with G:BOX chemiluminescence system using Syngene’s GeneSys software (Syngene; Cambridge, UK). Uncropped western blots scans were provided in the Source Data files.

Anderson A., Alfahad N., Wimalachandra D., Bouzinab K., Rudzinska P., Wood H., Fazey I., Xu H., Lyons T.J., Barnes N.M., Narendran P., Lord J.M., Rauz S., Ganley I.G., Curtis T.M., Wallace G.R, & Hombrebueno J.R. (2024). Relaxation of mitochondrial hyperfusion in the diabetic retina via N6-furfuryladenosine confers neuroprotection regardless of glycaemic status. Nature Communications, 15, 1124.

+ Open protocol

+ Expand

Immunoprecipitation of FLAG-RagB Complex

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

After the treatment, U2OS cells stably expressing FLAG-RagB and ER-E2F1 were washed twice with ice-cold PBS and incubated for 7 minutes at room temperature with 1 mM DSP crosslinker reagent in PBS (Thermo Scientific, USA). 1 M Tris-HCl (pH 7.5) was added 1:10 to quench DSP and cells were washed twice prior to lysis in ice-cold RIPA buffer (1% NP-40, 1% deoxycholate, 0.1%SDS) in the presence of protease and phosphatase inhibitors cocktails (Sigma Aldrich, USA). The soluble fractions from cell lysates were isolated by centrifugation at 13, 000 rpm for 5 minutes in a microfuge. For immunoprecipitations, 30 μl of a 33% slurry of anti-FLAG M2 beads (Sigma Aldrich, USA) was added to each lysate and incubated with rotation overnight at 4°C. Immunoprecipitates were washed three times with RIPA buffer supplemented with 500 mM NaCl and once with normal RIPA buffer. Immunoprecipitated proteins were resolved by 4%–20% SDS-PAGE.

Meo-Evoli N., Almacellas E., Massucci F.A., Gentilella A., Ambrosio S., Kozma S.C., Thomas G, & Tauler A. (2015). V-ATPase: a master effector of E2F1-mediated lysosomal trafficking, mTORC1 activation and autophagy. Oncotarget, 6(29), 28057-28070.

+ Open protocol

+ Expand

Protein Expression Analysis by Western Blot

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

Twenty four hours after cell seeding, the medium was replaced with the fresh one, and cells were incubated with previously indicated concentrations of inhibitors for 4 h. Cell lysates were harvested by addition of CB buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate) supplemented with protease and phosphatase inhibitors cocktails (Sigma). Protein concentration was determined with standard Bradford procedure (Sigma) (Bradford, 1976 (link)). Samples of an identical amount of protein (20 μg) were separated by 10% polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) according to Laemmli (Laemmli, 1970 (link)) and then transferred to nitrocellulose sheets, according to Towbin et al. (Towbin et al., 1979 (link)). Antibodies to Src, pSrc, GAPDH, as well as goat anti-mouse antibodies conjugated with horseradish peroxidase (Cell Signaling Technologies) were applied according to the manufacturer’s protocols. Immunoblots were developed using the Clarity Western ECL Substrate (Bio-Rad), scanned with ChemiDoc (Bio-Rad) and analyzed with ImageLab Software (ver. 6.0, Bio-Rad). At least three independent experiments were conducted.

Simiczyjew A., Pietraszek-Gremplewicz K., Dratkiewicz E., Podgórska M., Matkowski R., Ziętek M, & Nowak D. (2019). Combination of Selected MET and EGFR Inhibitors Decreases Melanoma Cells’ Invasive Abilities. Frontiers in Pharmacology, 10, 1116.

+ Open protocol

+ Expand

Western Blot Protein Quantification Protocol

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

The protocol applied in our previous research was utilized for this assay 16 (link), 17 (link). Briefly, the whole cellular protein was acquired by incubating cells in ice-cold RIPA lysis buffer (Beyotime Inc, NanTong, China) augmented with protease and phosphatase inhibitors cocktails (Sigma), per the manufacturer's protocol. The acquired proteins were isolated by SDS-PAGE, transplanted on the PVDF membranes, occluded for 1 h using 5% non-fat milk, rinsed, tagged at 4℃ with the indicated primary antibodies with gentle overnight shaking, and then labeled with horseradish peroxidase (HRP)-coupled secondary antibodies. The immunoreactive bands were visualized via enhanced chemiluminescence. The ECHS1 (1:100; #11305-1-AP) and β-actin (1:1000; #66009-1-Ig) antibodies were acquired from Proteintech and utilized in this investigation.

Lu T., Sun L., Fan Q., Yan J., Zhao D., Xu C, & Dong F. (2024). Expression and clinical significance of ECHS1 in gastric cancer. Journal of Cancer, 15(2), 418-427.

+ 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!