Proteases inhibitor cocktail

Manufactured by Roche

Sourced in Canada, United States, Germany

The Proteases inhibitor cocktail is a laboratory product designed to inhibit the activity of proteases, which are enzymes that break down proteins. It is a mixture of various chemical compounds that target and inhibit the function of different types of proteases. The core function of this product is to prevent the degradation of proteins during various experimental procedures, allowing researchers to study and analyze proteins more effectively.

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Lab products found in correlation

19 protocols using proteases inhibitor cocktail

Quantitative Analysis of Skin Inflammasome

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Protein from human and rodent skin was extracted in RIPA buffer containing phosphatases and proteases inhibitor cocktails (Roche). Protein concentrations were determined by the BCA protein assay kit (Pierce, Mississauga, ON, Canada). Proteins were resolved by SDS-PAGE followed by western blotting using the following antibodies at 1:1000 concentration: Caspase-1 (Cell Signaling, MA), cleaved Caspase-1 (Cell Signaling, MA), IL1β (Cell Signaling, MA), cleaved IL1β (Cell Signaling, MA), and GAPDH (Cell Signaling, MA). Species appropriate secondary antibodies conjugated to horseradish peroxidase (BioRad, Mississauga, ON, Canada) were used and proteins visualized by enhanced chemiluminescence using the BioRad ChemiDoc MP Imaging System. Band intensities were detected, normalized and quantified with the ChemiDoc and Image Lab 5.0 software (BioRad Laboratories, Hercules, CA). Antibody concentrations are expressed relative to GAPDH.

Vinaik R., Abdullahi A., Barayan D, & Jeschke M.G. (2019). NLRP3 Inflammasome Activity is Required for Wound Healing After Burns. Translational research : the journal of laboratory and clinical medicine, 217, 47-60.

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Western Blot Analysis of Inflammasome Proteins

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Proteins from rodent liver and adipose tissues were extracted in RIPA
buffer containing phosphatases and proteases inhibitor cocktails (Roche).
Protein concentrations were determined by the BCA protein assay kit (Pierce,
Mississauga, ON, Canada). Proteins were resolved by SDS-PAGE followed by western
blotting using the following antibodies at 1:500– 1:1000 concentration:
IL-1β (Cell Signaling, MA, USA), Caspase-1 (Abcam, MA USA), IL-18 (Abcam,
MA USA), ASC (Adipogen, CA, USA), NF-κB (Cell Signaling, MA, USA),
phospho- eIF2α (Cell Signaling, MA, USA), phospho-JNK (Cell Signaling,
MA, USA), BiP (Cell Signaling, MA, USA) and GAPDH (Cell Signaling, MA, USA).
Species appropriate secondary antibodies conjugated to horseradish peroxidise
(BioRad, Mississauga, ON, Canada) were used and proteins visualized by enhanced
chemiluminescence using the BioRad ChemiDoc MP Imaging System. Band intensities
were detected, normalized and quantified with the ChemiDoc and Image Lab 5.0
software (BioRad Laboratories, Hercules, CA). Antibody concentrations are
expressed relative to GAPDH. Due to inter-species variability between WT and
Nlrp3^−/− control
concentrations, all values are presented as a ratio relative to the mean
concentration of species- specific controls for a given antibody.

Satokata I., Tanaka K., Miura N., Miyamoto I., Satoh Y., Kondo S, & Okada Y. (1990). Characterization of a splicing mutation in group A xeroderma pigmentosum.. Proceedings of the National Academy of Sciences of the United States of America, 87(24), 9908-9912.

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Adipose Tissue Protein Analysis

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Proteins from rodent adipose tissue was extracted in RIPA buffer containing phosphatases and proteases inhibitor cocktails (Roche). Protein concentrations were determined by the BCA protein assay kit (Pierce, Mississauga, ON, Canada). Proteins were resolved by SDS-PAGE followed by western blotting using the following antibodies at 1:1000–1:5000 concentration: Fasn (Cell Signaling, MA) and GAPDH (Cell Signaling, MA, USA). Species appropriate secondary antibodies conjugated to horseradish peroxidase (BioRad, Mississauga, ON, Canada) were used and proteins visualized by enhanced chemiluminescence using the BioRad ChemiDoc MP Imaging System. Band intensities were detected, normalized and quantified with the ChemiDoc and Image Lab 5.0 software (BioRad Laboratories, Hercules, CA). Antibody concentrations are expressed relative to GAPDH. Due to inter-species variability between WT and NLRP3^−/− control concentrations, all values are presented as a ratio relative to the mean concentration of species-specific controls for a given antibody.

Vinaik R., Stanojcic M, & Jeschke M.G. (2018). NLRP3 Inflammasome Modulates Post-Burn Lipolysis and Hepatic Fat Infiltration via Fatty Acid Synthase. Scientific Reports, 8, 15197.

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Protein Extraction and Western Blot Analysis

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Protein samples derived from mouse skins and cells were extracted by RIPA lysis buffer containing phosphatases and proteases inhibitor cocktails (Roche, USA). Protein concentration was determined by BCA protein assay kit (Pierce, USA). For immunoblotting analysis, protein samples were subjected with SDS-polyacrylamide gel electrophoresis and proteins were transferred to PVDF membranes (Millipore). After being blocked, and incubated with primary antibodies and indicated HRP-coupled secondary antibody, membranes were visualized with ECL and images were captured using the Bio-Rad system. Band intensities were detected, normalized, and quantified with the Image Lab 5.0 software (Bio-Rad). The following antibodies were used in this research: NAT10 (Abcam, ab194297, 1:1000 dilution), STAT3 (CST, 30835, 1:1000 dilution), p-STAT3 (Abcam, ab76315, 1:1000 dilution), p-p65 (CST, 3033, 1:1000 dilution), p65 (CST, 8242, 1:1000 dilution), p-FAK(CST, 8556, 1:1000 dilution), FAK (Abcam, ab40794, 1:1000 dilution), Lamin B1 (Abcam, ab133741, 1:1000 dilution), poly-Ubiquitin (CST, 3936, 1:1000 dilution), Fibronectin(CST, 26836, 1:1000 dilution), α-SMA(Proteintech, 14395-1-AP, 1:1000 dilution), α-Tubulin (Beyotime, AT819, 1:5000 dilution), and GAPDH (Beyotime, AG019, 1:5000 dilution).

Wang B., Zhang J., Li G., Xu C., Yang L., Zhang J., Wu Y., Liu Y., Liu Z., Wang M., Li J., Tang X, & Liu B. (2023). N-acetyltransferase 10 promotes cutaneous wound repair via the NF-κB-IL-6 axis. Cell Death Discovery, 9, 324.

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Protein Expression Analysis in Skin Tissues

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Protein from human tissue, keloid tissue, and murine skin was extracted in RIPA buffer containing phosphatases and proteases inhibitor cocktails (Roche). Protein concentrations were determined by the BCA protein assay kit (Pierce). Proteins were resolved by SDS-PAGE followed by Western blotting using the following antibodies at 1:1000 concentration: cleaved caspase-1 (Cell Signaling Technology, D57A2), cleaved IL-1β (Cell Signaling Technology, D3A3Z), Glut1 (Abcam, ab15309), PKM2 (Cell Signaling Technology, 4053), Hif1α (Cell Signaling Technology, 36169), and GAPDH (Cell Signaling Technology, 5174). Species appropriate secondary antibodies conjugated to horseradish peroxidase (Bio-Rad) were used, and proteins were visualized by enhanced chemiluminescence using the Bio-Rad ChemiDoc MP Imaging System. Band intensities were detected, normalized, and quantified with the ChemiDoc and Image Lab 5.0 software (Bio-Rad). Western blot samples for identical markers and species were derived from the same experiment and were processed in parallel. Antibody concentrations are expressed relative to GAPDH.

Vinaik R., Barayan D., Auger C., Abdullahi A, & Jeschke M.G. (2020). Regulation of glycolysis and the Warburg effect in wound healing. JCI Insight, 5(17), e138949.

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Western Blot Analysis of Lung Proteins

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For in vivo, the lungs were removed and homogenized in RIPA buffer (Sigma-Aldrich/Merck, Darmstadt, Germany) containing proteases inhibitor cocktail (Roche, Basel, Switzerland). Protein was quantified by Bradford assay (Pierce^TM Coomassie [Bradford] Protein Assay) (Thermo Fisher Scientific, Waltham, MA, EUA). The supernatants were suspended with LDS Sample Buffer (4x) (Life Technologies). For in vitro, BMDM supernatants were denatured in loading buffer containing RIPA buffer, protease inhibitor, and LDS sample buffer (4x). Next, all samples were boiled and subjected to 4–12% SDS-polyacrylamide gel electrophoresis (PAGE) gel. The SDS-PAGE-separated proteins, transferred to nitrocellulose membranes, were immunoblotted with primary antibodies IL-1β (1:500; Sigma-Aldrich, St. Louis, MO, USA), caspase 1 p20 (1:500; clone 4B4, Genentech, San Francisco, CA, USA), caspase 8 (1:1000; cell singling #8592), and beta actin (1:5000; ab8227). Specific secondary antibodies were used afterward. Proteins in the blots were visualized by enhanced chemiluminescence using ECL (GE Healthcare). The data acquisition and bands were densitometrically quantified using iBright^TM CL1500 Imaging System (Thermo Fisher Scientific, Waltham, MA, EUA) or ImageJ software (V1.51) (Schneider et al., 2012 (link)), and the results were expressed as arbitrary units.

Souza C.O., Ketelut-Carneiro N., Milanezi C.M., Faccioli L.H., Gardinassi L.G, & Silva J.S. (2021). NLRC4 inhibits NLRP3 inflammasome and abrogates effective antifungal CD8+ T cell responses. iScience, 24(6), 102548.

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Cell Lysis and Western Blotting Protocol

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Cells were lysed in TEGN buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 420 mM NaCl, 10% glycerol, and 0.5% Nonidet P-40) containing proteases inhibitor cocktail (Roche) and 1 mM dithiothreitol (DTT). For Western blotting, the cell lysates were boiled in protein sample buffer (2 M β-mercaptoethanol, 12% sodium dodecyl sulfate (SDS), 0.5 M Tris, pH 6.8, 0.5 mg/mL bromophenol blue, and 30% glycerol), and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). Antibodies used were the following: LC3A/B antibody (#4108; Cell Signaling, Beverly, MA, USA), actin (A2066; Sigma), cleaved caspase-3 (#9661; Cell Signaling), PARP (#9542; Cell Signaling).

Wu C.C., Huang Y.F., Hsieh C.P., Chueh P.J, & Chen Y.L. (2016). Combined Use of Zoledronic Acid Augments Ursolic Acid-Induced Apoptosis in Human Osteosarcoma Cells through Enhanced Oxidative Stress and Autophagy. Molecules, 21(12), 1640.

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Western Blot Analysis of Alpha-Synuclein

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For Western blot analysis, cortex and olfactory bulb of mouse brains were homogenized in RIPA Buffer (50mM Tris/HCl pH7.4, 150 mM NaCl, 2 mM EDTA, 1% (v/v) NP-40, 0.1% (w/v) SDS) in the presence of a proteases inhibitor cocktail (Roche) with a douncer homogenizer Potter at 4°C. Protein levels in the tissue homogenates were quantified via BCA protein assay (Thermo Scientific). 80 μg of total proteins were mixed with the same volume of SDS sample buffer (0.125 M Tris/HCl pH 6.8, 4% SDS, 20% glycerol), separated on 15% SDS-PAGE, and blotted onto nitrocellulose membranes (Millipore). The blots were probed with a mouse anti-alpha-synuclein-1 antibody (1:2000, BD Transduction Laboratories) and a rabbit anti GAPDH antibody (1:2000, FL335, Santa Cruz Technology), followed by the incubation with secondary goat anti mouse and goat anti rabbit antibodies coupled to horseradish peroxidase (1:10000 Dianova, Hamburg, Germany). For the detection of proteins, membranes were incubated with the SuperSignal West Pico Sensitivity Substrate (Thermo Scientific) and visualized by VersaDoc gel imaging system (BioRad).

Schreglmann S.R., Regensburger M., Rockenstein E., Masliah E., Xiang W., Winkler J, & Winner B. (2015). The Temporal Expression Pattern of Alpha-Synuclein Modulates Olfactory Neurogenesis in Transgenic Mice. PLoS ONE, 10(5), e0126261.

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Dystrophin Immunoprecipitation from Cardiac Tissue

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Frozen hearts were pulverized and solubilized in homogenization buffer (1% Digitonin, 0.05% NP-40, 150 mM NaCl, and 50 mM Tris, pH 7.4) in the presence of proteases inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) [33 (link)]. The following procedures were performed at 4°C. Homogenates were solubilized by gentle shaking for 1 h, centrifuged at 16,100xg for 99 min and the supernatant was pre-cleared with protein G agarose beads (Cell Signaling, Danvers, MA) for 1h. Protein concentration of the homogenate was adjusted (0.8 μg protein/μl) and antibody against dystrophin (Abcam, ab15277) was added. Following an overnight incubation, protein G agarose beads were added for 1 h, washed in the homogenization buffer without digitonin and proteins were eluted by boiling with reducing sample buffer for western blot analysis. Control (data not shown) immunoprecipitations without addition of anti-dystrophin antibody or in the presence of unrelated antibody were performed to determine specificity of the interaction. Furthermore, each an immunoprecipitation reaction blot was probed for the presence of α-actinin, which was present in the homogenates but absent from the eluted fractions.

Strakova J., Dean J.D., Sharpe K., Meyers T.A., Odom G, & Townsend D. (2014). Dystrobrevin increases dystrophin's binding to the dystrophin-glycoprotein complex and provides protection during cardiac stress. Journal of molecular and cellular cardiology, 76, 106-115.

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Quantifying Immune Markers in Lung Samples

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IL-22, IL-17A, MPO, and AREG in BALF were determined by ELISA (R & D systems, Abingdon, UK), while IL-4, IL-5 in lung, and TGF-β1 from BAL were measured by Luminex immunoassay using MagPix reader (Bio-Rad) according to the manufacturer's instructions. Data were analyzed with Bio-Plex Manager software (Bio-Rad). Total protein levels in BALF were analyzed with the Bio-Rad DC Protein Assay. Collagen was measured with Soluble Collagen Assay Sircol™ (biocolor), according to the manufacturer's instructions on BAL and lung supernatants. For the protein measurement on the lung supernatant, the same part of the lung was harvested and gridded in 1 mL of PBS with proteases inhibitor cocktail (Roche). After centrifugation, supernatant was collected and frozen.

Michaudel C., Bataille F., Maillet I., Fauconnier L., Colas C., Sokol H., Straube M., Couturier-Maillard A., Dumoutier L., van Snick J., Quesniaux V.F., Togbe D, & Ryffel B. (2020). Ozone-Induced Aryl Hydrocarbon Receptor Activation Controls Lung Inflammation via Interleukin-22 Modulation. Frontiers in Immunology, 11, 144.

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