Protease inhibitor cocktail

Manufactured by GoldBio

Sourced in United States

Protease inhibitor cocktail is a laboratory product designed to inhibit the activity of proteases, which are enzymes that break down proteins. This cocktail contains a mixture of chemical compounds that work to prevent the degradation of proteins during various experimental procedures.

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Lab products found in correlation

Lds sample buffer, by Thermo Fisher Scientific (2 mentions) Phosphatase inhibitor cocktail, by Merck Group (2 mentions) Phosbind reagent, by Apexbio (2 mentions) Pvdf membrane, by Merck Group (1 mentions) Bradford assay, by Bio-Rad (1 mentions) Ripa buffer, by Merck Group (1 mentions) Versadoc imaging system, by Bio-Rad (1 mentions) Chemidoc xrs detection system, by Bio-Rad (1 mentions) β actin, by ZSGB-BIO (1 mentions) Hrp conjugated secondary antibody, by Bio-Rad (1 mentions) Peptide n glycosidase f, by New England Biolabs (1 mentions) Sp sepharose fast flow, by GE Healthcare (1 mentions) 2 8 3h adenine, by PerkinElmer (1 mentions) Lambda phosphatase, by New England Biolabs (1 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions) Chemidoc imaging system, by Bio-Rad (1 mentions) Hrp linked anti rabbit igg, by Cell Signaling Technology (1 mentions) Hrp linked anti mouse igg, by Cell Signaling Technology (1 mentions) Hrp linked anti rat igg, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

Protease inhibitors (6 citations) Protease and phosphatase inhibitor cocktails (2 citations) ProBlock™ Protease Inhibitor Cocktail-50 (2 citations) ProBlock gold protease inhibitor cocktail (2 citations)

7 protocols using protease inhibitor cocktail

Western Blot Analysis of Cellular Signaling

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Cells were harvested and prepared using RIPA buffer (Sigma-Aldrich, St. Louis, USA) supplemented with 1% protease inhibitor cocktail and 1% phenylmethanesulfonyl fluoride (Gold Biotechnology, USA) at 4°C and were collected by centrifugation. Protein concentrations were determined by the Bradford assay (Bio-Rad, Hercules, CA). Equal amounts of protein (10-40 µg) were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were washed and incubated at 4°C overnight with the following specific primary antibodies: rabbit polyclonal antibody against human TIPE (1:300; Boster, Wuhan, China), rabbit monoclonal antibody against VEGFR2 (1:1000; Abways, USA), rabbit monoclonal antibody against RAS (1:5000, Abcam, Kendall Square, Suite Cambridge, USA), PDK1, p-PDK1 (1:2000; Millipore, CA, USA), and β-actin (1:1000; ZSGB-Bio, Beijing, China). The next day, the membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and goat anti-rabbit IgG (1:2000; ZSGB-Bio, Beijing, China) antibodies for 1 hour at room temperature. Then, immune-reactive bands were visualized using a BIO-RAD ChemiDoc XRS+ Detection System (Bio-Rad, Hercules, CA) or the traditional darkroom method and quantified by densitometric analysis using a Versadoc imaging system (Bio-Rad, Hercules, CA).

Zhong M., Li N., Qiu X., Ye Y., Chen H., Hua J., Yin P, & Zhuang G. (2020). TIPE regulates VEGFR2 expression and promotes angiogenesis in colorectal cancer. International Journal of Biological Sciences, 16(2), 272-283.

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Phosphatase-Mediated Protein Dephosphorylation

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HEK293T cells from a near confluent 10 cm dish were harvested, pelleted by centrifugation, and resuspended in 500 μl lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% IGEPAL CA-630) supplemented with protease inhibitor cocktail (Goldbio, GB-331). Following incubation on ice for 15 minutes, extracts were cleared by centrifugation. Phosphatase reactions (100 μl total volume) containing cleared lysates supplemented with MnCl₂ (1 mM final concentration), 100 units lambda phosphatase (NEB, P0753S), and/or phosphatase inhibitor cocktail (2x final concentration) (Sigma, 4906837001) were prepared on ice and subsequently incubated at 30 °C for 1 hour. Reactions were then boiled with LDS sample buffer (Thermo Fisher, NP0008) supplemented with beta-mercaptoethanol, resolved by SDS-PAGE using a 6% acrylamide gel supplemented with 75 μM PhosBind reagent (APExBIO, F4002) and 150 μM MnCl₂, and transferred to nitrocellulose membrane for immunoblotting.

Acharya A., Bret H., Huang J.W., Mütze M., Göse M., Kissling V., Seidel R., Ciccia A., Guérois R, & Cejka P. (2023). Mechanism of DNA unwinding by hexameric MCM8–9 in complex with HROB. Research Square.

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HEK293 Cell Lysis for Proteasome Inhibition

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HEK293 cells were grown in media containing DMEM, 10% (v/v) FBS, 1× glutamax, 100 U/mL penicillin, and 100 mg/mL streptomycin, at 37 °C, 5% CO₂. Once cells reached 80–90% confluency, they were washed with cold PBS, and treated with either DMSO, 10 µM MG132, 30 µM PR619, or combination of 10 µM MG132 and 30 µM PR619 for 4 h. Cells were then harvested by first removing the growth media, then scrapping cells in the presence of cold PBS (2 mL). Cells were pelleted at 1000g, 4 °C for 5 min and the supernatant decanted. Cell pellets were resuspended in cold PBS (10 mL) and pelleted again at 1000g, 4 °C for 10 min. To every 10× 150 mm plates of HEK cells, was added 5 mL of cold lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 0.05% IGEPAL CA-630, 10 mM 2-chloroacetamide, 10 mM N-ethylmaleamide, and 1× protease inhibitor cocktail (Gold Biotechnology)). In addition, lysis buffer was supplemented with 10 µM MG132, 30 µM PR619, or both. Cells were incubated in lysis buffer on ice for 30 min prior to sonication. The resulting lysates were clarified at 16000g, 4 °C for 30 min.

Rana A.S., Ge Y, & Strieter E.R. (2017). Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry (UbiChEM-MS) Reveals Cell-Cycle Dependent Formation of Lys11/Lys48 Branched Ubiquitin Chains. Journal of proteome research, 16(9), 3363-3369.

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Investigating GPCR Regulation by Kinases

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Cell culture reagents are from Mediatech (Herdon, VA). Peptide N-glycosidase F was from New England Biolabs (Beverly, MA). Polyclonal primary antibodies to pS-(355,356) C-Tail of the β2AR, and to GRK2, GRK5, and GRK6 are from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody 2G3 for pS-(262) of the β2AR was as described (Tran et al. 2004 (link)). N-terminal 6 His-tagged, recombinant, full-length human GRK6, and GRK7 were purchased from Millipore (Dundee, UK). Human His₆-tagged GRK5 was a kind gift from John Tesmer (University of Michigan, Ann Arbor, MI). The HRP-conjugated secondary antibody was from BioRad. Enhanced chemiluminescence SuperSignal reagent was purchased from Thermo Scientific (Rockford, IL), and blue X-ray film was from Phenix (Candler, NC). SP-Sepharose Fast Flow was purchased from GE Healthcare (Piscataway, NJ). Sf-900 II SFM insect cell culture medium was purchased from Gibco (Grand Island, NY). Purified tubulin from bovine brain was purchased from Cytoskeleton (Denver, CO). [2,8-³H]adenine was purchased from Perkin Elmer. Protease inhibitor cocktail was from Gold Biotechnology (St. Louis, MO).

Baameur F., Hammitt R.A., Friedman J., McMurray J.S, & Clark R.B. (2014). Biochemical and Cellular Specificity of Peptide Inhibitors of G Protein-Coupled Receptor Kinases. International journal of peptide research and therapeutics, 20(1), 1-12.

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Expression and Purification of PDF-E133A

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Wild-type PDF, MAP, MAP-H79A, SRP and TF were expressed and purified as described [23 (link),74 (link),75 (link)].
To construct the expression plasmid for PDF-E133A, an 11-residue ybbR tag was fused to the protein coding sequence, and subcloned into pET28a vector containing a cleavable N-terminal His₆-SUMO tag [34 (link)]. To express PDF-E133A, BL21 star (DE3) cells were induced at OD = 0.6 with 0.5 mM IPTG at 30 °C for 3 h, and lysed by sonication in buffer A (20 mM Hepes-KOH, 300 mM NaCl, 0.2 mM CoCl₂, 10% glycerol, 1 mM TCEP, pH 7.5) containing protease inhibitor cocktail (GoldBio). Clarified lysates were purified with Ni-NTA resin, and the His₆-SUMO tag was removed by overnight dialysis in the presence of His₆-Ulp1 protease against buffer B (20 mM Hepes-KOH, 20 mM NaCl, 10% glycerol, pH 7.5). Tag-less proteins were further purified over a Ni-NTA column, supplemented with 100 mM NaCl, 0.2 mM CoCl₂, 1 mM TCEP, and 50% glycerol, and stored in –30 °C.

Yang C.I., Kim J, & Shan S.O. (2022). Ribosome-nascent chain interaction regulates N-terminal protein modification. Journal of molecular biology, 434(9), 167535.

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Phosphatase Activity Assay in HEK293T

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HEK293T cells from a near-confluent 10 cm dish were harvested, pelleted by centrifugation and resuspended in 500 µl lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% IGEPAL CA-630) supplemented with protease inhibitor cocktail (Goldbio, GB-331). Following incubation on ice for 15 minutes, extracts were cleared by centrifugation. Phosphatase reactions (100 µl total volume) containing cleared lysates supplemented with MnCl₂ (1 mM final concentration), 100 units lambda phosphatase (NEB, P0753S), and/or phosphatase inhibitor cocktail (2x final concentration) (Sigma, 4906837001) were prepared on ice and subsequently incubated at 30 °C for 1 hour. Reactions were then boiled with LDS sample buffer (Thermo Fisher, NP0008) supplemented with β-mercaptoethanol, resolved by SDS-PAGE using a 6% acrylamide gel supplemented with 75 µM PhosBind reagent (APExBIO, F4002) and 150 µM MnCl₂, and transferred to nitrocellulose membrane for immunoblotting.

Acharya A., Bret H., Huang J.W., Mütze M., Göse M., Kissling V.M., Seidel R., Ciccia A., Guérois R, & Cejka P. (2024). Mechanism of DNA unwinding by MCM8-9 in complex with HROB. Nature Communications, 15, 3584.

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Western Blot Analysis of Brain Tissue

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The cortex and hippocampus were collected from non-perfused brain collected during sacrifice and were immediately frozen in liquid nitrogen and stored at −80 °C until use. For western blotting, 15 mg tissues were homogenized in RIPA lysis buffer (ThermoFisher Scientific, Waltham, MA) containing phosphatase and protease inhibitor cocktail (Goldbio Cat. No. GB-108-2). Western blotting was performed using 40 μg protein/well as previously described (Mohammed et al. 2021). Primary antibodies against the following proteins were used: Phospho-MLKL (Ser345) (Abcam, Cat. No. ab196436), MLKL (Millipore, Cat. No. MABC604), β-tubulin (Cat. No. T5201), and GAPDH (Cat. No. G8795) were purchased from Sigma-Aldrich (St. Louis, MO). HRP-linked anti-rabbit IgG, HRP-linked anti-mouse IgG, or HRP-linked anti-rat IgG were used as secondary antibodies (Cell Signaling Technology, Danvers, MA). Membranes were imaged with ChemiDoc imaging system (Bio-Rad) and quantified using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA).

Thadathil N., Nicklas E.H., Mohammed S., Lewis TL J.r., Richardson A, & Deepa S.S. (2021). Necroptosis increases with age in the brain and contributes to age-related neuroinflammation. GeroScience, 43(5), 2345-2361.

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