Protease inhibitor

Manufactured by Bio-Rad

Sourced in United States, Germany

Protease inhibitors are chemical compounds used in laboratory settings to prevent the breakdown of proteins by proteolytic enzymes. They function by selectively binding to and inactivating specific proteases, thereby preserving the integrity of protein samples during various experimental procedures.

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Lab products found in correlation

Bradford assay, by Bio-Rad (3 mentions) Sds page, by Bio-Rad (2 mentions) Trans blot turbo transfer system, by Bio-Rad (2 mentions) Ripa buffer, by Bio-Rad (2 mentions) Protease inhibitor, by Merck Group (2 mentions) Clarity western ecl substrate, by Bio-Rad (2 mentions) T5168, by Merck Group (2 mentions) Protease inhibitor, by Roche (2 mentions) Image lab 4, by Bio-Rad (2 mentions) Abcg1, by Santa Cruz Biotechnology (1 mentions) Sr b1, by Santa Cruz Biotechnology (1 mentions) Microbca protein assay, by Bio-Rad (1 mentions) Gs 800 calibrated densitometer, by Bio-Rad (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Ripa buffer, by Merck Group (1 mentions) β actin, by Merck Group (1 mentions) Srebp1, by Abcam (1 mentions) Abca1, by Abcam (1 mentions) Chemidoc system, by Bio-Rad (1 mentions) Staurosporine, by Merck Group (1 mentions)

Spelling variants of this product

Protease inhibitor cocktail (28 citations) Complete protease inhibitor cocktail (6 citations) Halt™ Protease Inhibitor Cocktail (3 citations) Phosphatase and protease inhibitors (2 citations) Halt Protease and Phosphatase Inhibitor Cocktail (2 citations)

16 protocols using protease inhibitor

Quantification of Nuclear Protein Levels

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BMDM were lysed in RIPA buffer (Sigma-Aldrich) containing protease inhibitors (Bio-Rad, Hercules, CA) for whole cell protein extraction. Nuclear protein extraction was performed as previously described [24 (link)]. Briefly, cells were resuspended in a fractionation buffer containing protease inhibitors (Bio-Rad), passed through a 22.5 gauge needle 30 times, and centrifuged at 1000 × g for 7 min at 4°C. The pellet was resuspended in another fractionation buffer containing protease inhibitors (Bio-Rad), rotated at 4°C for 1 hr, and centrifuged at 100,000 × g for 30 min at 4°C in a Beckman TLA 100.2 rotor. The supernatant from this spin was designated the nuclear extract.
Whole cell and nuclear protein concentrations were determined by MicroBCA protein assay (Bio-Rad). Proteins were separated by SDS-PAGE (Bio-Rad) under reducing conditions and transferred to nitrocellulose membranes (Bio-Rad). Quantification by Western blotting was performed using the following primary antibodies: ABCA1 (Abcam, Cambridge, MA), ABCG1 (Santa Cruz Biotechnology, Santa Cruz, CA), SR-B1 (Santa Cruz), LXR-α (Abcam), SREBP-1 (Abcam), and β-actin (Sigma-Aldrich). Secondary antibodies were purchased from Santa Cruz Biotechnology. Signals were visualized by chemiluminescence (Amersham Biosciences, Piscataway, NJ) and quantified using a GS-800 calibrated densitometer (Bio-Rad).

Spartano N.L., Lamon-Fava S., Matthan N.R., Obin M.S., Greenberg A.S, & Lichtenstein A.H. (2014). Linoleic acid suppresses cholesterol efflux and ATP-binding cassette transporters in murine bone marrow-derived macrophages. Lipids, 49(5), 415-422.

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Western Blot Analysis of Protein Lysates

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Total protein lysates were isolated from growing cells using 1x radioimmunoprecipitation assay buffer [RIPA, 1% (v/v) NP40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS in 1x PBS buffer] with freshly added protease inhibitors (cocktail from Bio-Rad Laboratories, Hercules, CA). Total protein (10–30 μg) was separated by 4–20% (w/v) gradient polyacrylamide gels and transferred onto nitrocellulose membranes using the Trans-Blot Turbo transfer system (all Bio-Rad Laboratories). Membranes were blocked overnight at 4 °C in 1x TBS containing 5% (w/v) nonfat milk and 1% (w/v) bovine serum albumin, and then incubated with the following antibodies: anti-P-gp (clone C219, EMD Millipore, Billerica, MA), and specific antibodies for total Syk (D3Z1E), p-Syk (Tyr525/526), and GAPDH (all Cell Signaling Technology, Danvers, MA). These primary antibodies were recognized by species-appropriate horseradish peroxidase-conjugated secondary antibodies, and detected using the Clarity Western ECL substrate on a ChemiDoc System (Bio-Rad).

Duran G.E, & Sikic B.I. (2019). The Syk inhibitor R406 is a modulator of P-glycoprotein (ABCB1)-mediated multidrug resistance. PLoS ONE, 14(1), e0210879.

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Protein Extraction and Quantification

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Tissue samples for protein assays were quickly frozen in liquid nitrogen and pulverized with a mortar and pestle. Tissue powder was added to 1.5 mL Eppendorf tubes and solubilized in RIPA buffer containing protease inhibitors (Bio-Rad, Hercules, CA, USA). 30–80 μg of total protein based on the measurements using a Bradford assay (Bio-Rad, Hercules, CA, USA) were visualized using 10% SDS-PAGE minigels (Jiancheng Bioengineering Institute, Nanjing, China). The gels were transferred onto nitrocellulose membranes using standard procedures [19 (link)]. Gel bands were scanned and quantified with the Gelpro 3.0 software (Jiancheng Bioengineering Institute, Nanjing, China).

Wu S., Wang Q., Wang J., Duan B., Tang Q., Sun Z., Han J., Shan C., Wang Z, & Hao Z. (2020). Protocatechuic aldehyde from Salvia miltiorrhiza exhibits an anti-inflammatory effect through inhibiting MAPK signalling pathway. BMC Complementary Medicine and Therapies, 20, 347.

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Podocyte Knockdown Response Profiling

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Mouse podocytes were cultured as described. At day 10, cells were exposed to a pool of on-target plus small interfering RNA (siRNA) for GLUT4, GLUT1, AMPKα, and TSC1 (Dharmacon, Lafayette, CO), and on-target plus nontargeting siRNA. After 6, 12, 24, and 48 h, cells were collected in lysis buffer in the presence of protease inhibitors (Bio-Rad) for protein analysis and in sample buffer for the analysis of apoptosis, and were fixed and stained with phalloidin to study the morphology of the actin cytoskeleton. Images were acquired by confocal microscopy as well as by light microscopy. Annexin V (Vybrant Apoptosis Assay; Invitrogen) staining was used to study apoptosis and quantitative assessment performed by flow cytometry with BD Diva 6.0 Software (BD LSRII System; BD Biosciences, San Jose, CA). Staurosporine (1 mmol/L for 2 h; Sigma) was used as a positive control.

Guzman J., Jauregui A.N., Merscher-Gomez S., Maiguel D., Muresan C., Mitrofanova A., Diez-Sampedro A., Szust J., Yoo T.H., Villarreal R., Pedigo C., Molano R.D., Johnson K., Kahn B., Hartleben B., Huber T.B., Saha J., Burke GW I.I.I., Abel E.D., Brosius F.C, & Fornoni A. (2014). Podocyte-Specific GLUT4-Deficient Mice Have Fewer and Larger Podocytes and Are Protected From Diabetic Nephropathy. Diabetes, 63(2), 701-714.

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Multimeric Protein Complex Purification

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Dam1c, MIND, and all MIND mutants were expressed in Escherichia coli from polycistronic vectors. Ndc80c was expressed from two bicistronic vectors encoding Ndc80/Nuf2 and Spc24/Spc25 (Wei et al., 2005 (link)). The protein subcomplexes purified as previously described using either a His₆- or FLAG- affinity tag followed by size exclusion chromatography (SEC)(Gestaut et al., 2008 (link); Gestaut et al., 2010 (link); Kudalkar et al., 2015 (link)).
Briefly, His₆-tagged protein subcomplexes were expressed from polycistronic pST39 vectors in BL21 cells and induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 16 hr at 18°C. Cells were lysed with a French press and the subcomplex was immobilized on a Ni-charged IMAC resin column (Bio-Rad) in either MIND buffer (50 mM sodium phosphate buffer, pH 7.0, 200 mM NaCl), Ndc80c buffer (50 mM HEPES buffer, pH 7.6, 300 mM NaCl), or Dam1c buffer (50 mM sodium phosphate buffer, pH 6.9, 500 mM NaCl) supplemented with protease inhibitors (Roche), 5 mM imidazole, and 1 mM PMSF. The resin was then washed, and subcomplex eluted with buffer containing 300 mM imidazole before being further purified using a Superdex 200 size exclusion column (GE Healthcare) in either MIND buffer, Ndc80c buffer with 200 mM NaCl, or Dam1c buffer.

Hamilton G.E., Helgeson L.A., Noland C.L., Asbury C.L., Dimitrova Y.N, & Davis T.N. (2020). Reconstitution reveals two paths of force transmission through the kinetochore. eLife, 9, e56582.

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Immunoprecipitation of P-cadherin and β4 integrin

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BT-20 cells grown in monolayer were lysed with PBS containing 1% NP40 (Sigma-Aldrich) and 2 mM calcium chloride (Sigma-Aldrich), with phosphatase (Sigma-Aldrich) and protease inhibitors (Roche Diagnostics Gmbh). 500μg of cell lysate was precleared with Protein G magnetic beads (Millipore, Temecula, CA) for 10 minutes at room temperature and then incubated overnight at 4°C with 2 μg of mouse monoclonal anti-P-cadherin (Abcam, Cambridge, UK) or rabbit polyclonal anti-β4 integrin (Santa Cruz Biotechnology) or its corresponding control isotype (IgG1 mouse or IgG1 rabbit, company, respectively). The samples were then incubated with the Protein G magnetic beads (Millipore) for 10 minutes at room temperature. The beads were washed three times with washing buffer (lysis buffer diluted 1:5, containing phosphatase and protease inhibitors, as stated above) and boiled for 5 minutes in Laemmli buffer with β-mercaptoethanol (BioRad, Hercules, CA). Samples were subjected to SDS-PAGE and immunoblotting as previously described.

Vieira A.F., Ribeiro A.S., Dionísio M.R., Sousa B., Nobre A.R., Albergaria A., Santiago-Gómez A., Mendes N., Gerhard R., Schmitt F., Clarke R.B, & Paredes J. (2014). P-cadherin signals through the laminin receptor α6β4 integrin to induce stem cell and invasive properties in basal-like breast cancer cells. Oncotarget, 5(3), 679-692.

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Western Blot Analysis of Key Proteins

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Total protein lysates were isolated from growing cells using 1x radioimmunoprecipitation assay buffer [RIPA, 1% (v/v) NP40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS in 1x PBS buffer] with freshly added protease inhibitors (cocktail from Bio-Rad Laboratories, Hercules, CA). Total protein (10–25 μg) was separated by 4–20% (w/v) gradient polyacrylamide gels and transferred onto nitrocellulose membranes using the Trans-Blot Turbo transfer system (all Bio-Rad Laboratories). Membranes were blocked overnight at 4 °C in 1x TBS containing 5% (w/v) nonfat milk and 1% (w/v) bovine serum albumin, and then incubated with the following antibodies: anti-P-gp, BCRP, and MRP2 (Signet Laboratories, Dedham, MA), anti-MRP7 (Thermo Fisher Scientific, Rockford, IL), anti-class I, pan α- and β-tubulin (Sigma Aldrich), anti-class II and III β-tubulin (Covance, Berkeley, CA), class IV β-tubulin (Abcam, Cambridge, MA), anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA), and specific antibodies for BRCA1, Bcl2, inhibitors of apoptosis, and p21 (Cell Signaling Technology, Danvers, MA). These primary antibodies were recognized by species-appropriate horseradish peroxidase-conjugated secondary antibodies, and detected using the Clarity Western ECL substrate (Bio-Rad).

Duran G.E., Wang Y.C., Francisco E.B., Rose J.C., Martinez F.J., Coller J., Brassard D., Vrignaud P, & Sikic B.I. (2014). Mechanisms of Resistance to Cabazitaxel. Molecular cancer therapeutics, 14(1), 193-201.

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Immunoprecipitation of GFP-tagged proteins

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~1,200 N-GFP (N^55e11 ; N-GFP) or Sca-GFP (sca^5-120 > UAS-ScaGFP) heads were homogenized in ice cold co-IP lysis buffer (50 mM HEPES (pH 7.2), 150 mM NaCL, 2 mM MgCl₂, 2 mM CaCl₂, 5 mM KCl, 0.1% Igepal, 20% glycerol) with protease inhibitors (Sigma I3911). Insoluble material was pelleted by centrifugation at 5,000 rpm for 5 min. Supernatants were incubated with either rabbit αGFP or mock IgG for 2 hr. Protein-bound antibody was collected by incubating with 10 uL of Dynabeads Protein A for 1 hr and beads were collected with a magnet. Protein-bound beads were gently washed once with co-IP lysis buffer with no Igepal or protease inhibitors, then eluted with 2× Laemmli sample buffer (Bio-Rad). Samples were reduced with 1% beta-mercaptoethanol and boiled for 5 min. Samples used for Scabrous blots were not reduced, and were incubated with 10 mM N-ethylmaleimide (NEM) to prevent artificial dimerization.

Petruccelli E., Feyder M., Ledru N., Jaques Y., Anderson E, & Kaun K.R. (2018). Alcohol activates Scabrous/Notch to influence associated memories. Neuron, 100(5), 1209-1223.e4.

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Purification of Human Topoisomerase II Isoforms

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Human TOP1 was purified from baculovirus as described (23 (link)). Human TOP2α was purified from yeast strains JEL1 top1Δ transformed with 12-URA-B 6×His-hTOP2α. Induction of TOP2 by galactose as described (24 (link)). Yeast cells were lysed in equilibration buffer (300 mM KCl, 10 mM imidazole, 20mM Tris HCl pH 7.7, 10% glycerol and protease inhibitor cocktail (Sigma Aldrich, Cat# P8215)) by glass bead homogenization. Lysates were incubated with Ni-NTA resin and washed using wash buffer #1 (300 mM KCL, 30 mM imidazole, 20M Tris HCl pH 7.7, 10% glycerol and protease inhibitors) then wash buffer #2 (150 mM KCl, 30 mM imidazole, 20 mM Tris HCl pH 7.7, 10% glycerol and protease inhibitor cocktail). hTOP2α and β were eluted on a Poly-Prep chromatography column (Bio-Rad, Cat# 7311550) with elution buffer (150 mM KCl, 300 mM imidazole, 20 mM Tris HCl pH 7.7, 10% glycerol and protease inhibitors). The peak protein fractions were dialyzed in dialysis buffer (750mM KCl, 50 mM Tris HCl pH 7.7, 20% glycerol, 0.1 mM EDTA and 0.5mM DTT) and His tag was removed using TEV protease

Marzi L., Sun Y., Huang S.Y., James A., Difilippantonio S, & Pommier Y. (2020). The indenoisoquinoline LMP517: a novel antitumor agent targeting both TOP1 and TOP2. Molecular cancer therapeutics, 19(8), 1589-1597.

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Protein Expression Analysis in Kidney Samples

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Proteins were extracted from kidney tissue samples by RIPA buffer containing a cocktail of protease inhibitors (Bio-Rad Inc.). Protein was quantified using a Bradford assay kit (SK3041; Bio Basic Inc., Markham Ontario L3R 8T4 Canada), then separated by 10% SDS-PAGE (Bio-Rad Laboratories Inc. Cat#161-0181), and transferred to PDVF membranes. Membranes were immunoblotted with indicated primary antibodies prepared at 5% blocking buffer (1 : 1000) and were cultured overnight at 4°C. After extensive washing in TBS buffer, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies (Goat anti-rabbit IgG-HRP-1 mg Goat mab-Novus Biologicals) and developed using the chemiluminescent substrate (ClarityTM Western ECL substrate Bio-Rad Cat#170-5060). β-Actin was used as a loading control. Image analysis software was assessed by normalization procedure of each phosphorylated active target protein versus corresponding control sample total protein on the ChemiDoc MP imager.
Primary antibodies contained antibody t-PI3K (Cat#3358), p-PI3K (sc-1637), t-Akt (Cat#9272), p-Akt (Cat#9271), t-p65 (Cat#3034), p-p65 (Cat#3033,), t-NF-κB inhibitor α (IκBα, Cat#9242), p-IκBα (Cat#2859), cleaved-caspase-3 (Cat#AB3623), and β-actin (sc-8432).

Yassin N.Y., AbouZid S.F., El-Kalaawy A.M., Ali T.M., Elesawy B.H, & Ahmed O.M. (2021). Tackling of Renal Carcinogenesis in Wistar Rats by Silybum marianum Total Extract, Silymarin, and Silibinin via Modulation of Oxidative Stress, Apoptosis, Nrf2, PPARγ, NF-κB, and PI3K/Akt Signaling Pathways. Oxidative Medicine and Cellular Longevity, 2021, 7665169.

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