Protease inhibitor

Manufactured by G Biosciences

Sourced in United States

Protease inhibitor is a type of lab equipment used to inhibit the activity of proteases, which are enzymes that break down proteins. It is a vital tool for researchers studying protein function, stability, and regulation.

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Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Phosphatase inhibitor, by Merck Group (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Bovine serum albumin (bsa), by MP Biomedicals (2 mentions) Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions) C digit, by LI COR (1 mentions) Pvdf membrane, by Merck Group (1 mentions) G box, by Syngene (1 mentions) Rab buffer, by G Biosciences (1 mentions) Optima tlx, by Beckman Coulter (1 mentions) Polytron immersion disperser homogenizer, by Kinematica (1 mentions) Triethylammonium bicarbonate, by Merck Group (1 mentions) Trifluoroacetic acid, by Fujifilm (1 mentions) Ammonium hydroxide solution, by Merck Group (1 mentions) Cellytic mt cell lysis reagent, by Merck Group (1 mentions) Bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Tween 20, by Merck Group (1 mentions) Formic acid, by Merck Group (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Trypsin, by Promega (1 mentions)

Spelling variants of this product

Protease inhibitor cocktail (16 citations) ProteaseArrest protease inhibitors (8 citations) ProteaseArrest protease inhibitor cocktail (6 citations) Protease and phosphatase inhibitors (4 citations)

11 protocols using protease inhibitor

Nuclear Protein Extraction Protocol

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To examine the effect of FA on nuclear translocation of NFκB, the NE-PER® nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, Rockford, IL) were used and the extraction was performed according to the manufacturer’s protocol. Briefly, the cell pellet was disrupted in cytoplasmic extraction reagent I buffer plus protease inhibitor (G-Biosciences), and then lysed in cytoplasmic extraction reagent II buffer to release cytoplasmic proteins. After being centrifuged at 13,000 rpm for 10 min at 4 °C, the insoluble pellet, which contained nuclear proteins, was washed with ice-cold PBS twice, and then incubated in nuclear extraction reagent buffer plus protease inhibitor (G-Biosciences).

Kuo C.T., Chang C, & Lee W.S. (2015). Folic acid inhibits COLO-205 colon cancer cell proliferation through activating the FRα/c-SRC/ERK1/2/NFκB/TP53 pathway: in vitro and in vivo studies. Scientific Reports, 5, 11187.

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Synaptosomal Fractionation from Hippocampus

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Crude synaptosomal fractions were isolated from dissected left hippocampal brain tissues (n = 4/genotype and age) by homogenizing the tissue with a 2 cm³ glass-Teflon homogenizer at 800 rpm in lysis buffer (0.32 M sucrose, 5 mM Hepes, and 10 ml ddH₂O) supplemented with 1:100 diluted protease inhibitors (G-Biosciences, Cat. no: 786-433) and 1:100 diluted phosphatase inhibitors (Sigma–Aldrich, Cat. no: P0044) in a proportion of 1:10 (mg tissue/µl buffer). The homogenates were centrifuged at 1000 g at 4 °C for 10 min to remove nuclei and debris, and the supernatant was centrifuged again at 12,000 g at 4 °C for 20 min. Supernatant composed of the light membrane fraction and soluble proteins (S2) was transferred to a new tube, and the pellet containing crude synaptosomes and mitochondria (P2) was resuspended in 75 μl RIPA buffer supplemented with 1:100 diluted protease inhibitors and 1:100 diluted phosphatase inhibitors. The protein concentration was determined by Pierce™ BCA Protein Assay Kit (Thermo Fisher Sci., Cat. no: 23225).

Naia L., Shimozawa M., Bereczki E., Li X., Liu J., Jiang R., Giraud R., Leal N.S., Pinho C.M., Berger E., Falk V.L., Dentoni G., Ankarcrona M, & Nilsson P. (2023). Mitochondrial hypermetabolism precedes impaired autophagy and synaptic disorganization in App knock-in Alzheimer mouse models. Molecular Psychiatry, 28(9), 3966-3981.

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Western Blot Protein Extraction Protocol

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Cells grown on 10 cm, 6 well or 12 well culture dishes were scraped and washed once with 1 × phosphate buffered saline, pH 7.4 and centrifuged for 3 min at 1500 × g. Cell pellets were lysed using RIPA lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (G-Biosciences). After incubation for 30 min on ice, extracts were centrifuged at 20,000 × g for 10 min at 4 °C. The supernatant was collected as the whole cell lysates. Protein concentrations were determined with Bradford assay when necessary. Aliquots of total extracts were incubated with sample buffer containing 100 mM DTT at 37 °C for 10 min. Samples were separated by SDS-PAGE. Proteins were transferred to PVDF membranes (Millipore) followed by immunoblotting. ECL detection was performed according to manufacturer's instructions (Millipore) and blots were imaged with G-Box (Syngene) or C-DiGit (Li-COR). C-DiGit software was used for quantification. Human tissue lysates megablot for western was purchased from BioChain. For medium samples, during transfection/mock transfection the cells were grown on OPTI-MEM. Medium samples were collected 24 h post transfection and concentrated with spin column (Pierce concentrator, 10 K MWCO) before proceeding to protein gels.

De Silva B., Adams J, & Lee S.Y. (2015). Proteolytic processing of the neuronal ceroid lipofuscinosis related lysosomal protein CLN5. Experimental cell research, 338(1), 45-53.

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Fractionation of Mouse Hippocampal Proteins

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The hippocampus was dissected from snap frozen mouse hemi-brains after thawing on ice. The hippocampal tissue was weighed and homogenized using a Polytron immersion disperser homogenizer (Kinematica AG) in ice-cold RAB buffer (G Biosciences) at 10 µl mg⁻¹ tissue, supplemented by phosphatase inhibitors (Roche) and protease inhibitors (Roche). Samples were then centrifuged using an Optima TLX ultracentrifuge (Beckman Coulter) at 50,000g for 20 min at 4 °C and the supernatant was collected as the RAB-soluble fraction. The pellets were resuspended in ice-cold RIPA buffer (Thermo Scientific) at 10 µl mg⁻¹ tissue and centrifuged at 50,000g for 20 min at 4 °C. The supernatant was collected as the RIPA-soluble fraction and the pellet was stored at −80 °C for further use. All fractions were stored at −80 °C until further analyses.

Koutsodendris N., Blumenfeld J., Agrawal A., Traglia M., Grone B., Zilberter M., Yip O., Rao A., Nelson M.R., Hao Y., Thomas R., Yoon S.Y., Arriola P, & Huang Y. (2023). Neuronal APOE4 removal protects against tau-mediated gliosis, neurodegeneration and myelin deficits. Nature Aging, 3(3), 275-296.

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Protein Extraction and Quantification

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CelLytic™ MT cell lysis reagent, ammonium hydroxide solution, formic acid (FA), triethylammonium bicarbonate (TEABC), and Tween-20 were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Trifluoroacetic acid was purchased from Wako (Osaka, Japan). Clarity™ Western ECL Substrate and nitrocellulose membranes were purchased from Bio-Rad (Hercules, CA, USA). Acetonitrile (ACN) was purchased from Spectrum (California, USA). Ethylenediaminetetraacetic acid (EDTA) and protease inhibitors were purchased from G-Biosciences (St. Louis, MO, USA). Trypsin was purchased from Promega (Madison, USA). The bicinchoninic acid (BCA) protein assay kit and TMT assay were purchased from Thermo Fisher Scientific (Rockford, USA). Furthermore, 10% neutral buffered formalin was purchased from CHIN IPAO CO., LTD (Taipei, Taiwan), paraffin was purchased from Leica Microsystems Pvt. Ltd. (Macquarie Park, Australia), and haematoxylin and eosin (H&E) were purchased from Roche Diagnostics (Indianapolis, USA).

Jheng Y.T., Putri D.U., Chuang H.C., Lee K.Y., Chou H.C., Wang S.Y, & Han C.L. (2021). Prolonged exposure to traffic-related particulate matter and gaseous pollutants implicate distinct molecular mechanisms of lung injury in rats. Particle and Fibre Toxicology, 18, 24.

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Hippocampal Protein Extraction Protocol

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Right hippocampi from the same individuals used for RNA-seq experiments (n = 3/genotype and age) were cut in pieces while kept cold on ice and homogenized with a 2 cm³ glass-Teflon homogenizer in RIPA buffer (150 mM NaCl, 50 mM Tris, 1% Triton X-100, 0.5% DOC, 0.1% SDS; pH = 7.5) supplemented with 1:100 protease inhibitors (G-Biosciences, Cat. no: 786-433) and 1:100 phosphatase inhibitors (Sigma–Aldrich, Cat. no: P0044) in a proportion of 1:15 (mg tissue/µl buffer). The homogenates were then sonicated for 15 s on ice and centrifuged at 20,800 g for 30 min, at 4 °C, to remove cell debris. Supernatant was collected and protein concentration determined by Pierce™ BCA Protein Assay Kit (Thermo Fisher Sci., Cat. no: 23225).

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Mitochondrial Protein Purification

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Mitochondrial pallets were lysed in PBS containing 1% Triton X100 (Sigma), protease inhibitor (G Biosciences) and 10 mM imidazole (GE Healthcare). The lysate was cleared via centrifugation and loaded to His Spin Trap spin column (GE Healthcare). The column was sequentially washed with PBC containing 20, 40, and 80 mM imidazole and eluted with PBS containing 200 mM imidazole. 100 mM DTT was included in the lysis buffer when indicated.

Chang H.C., Shapiro J.S., Jiang X., Senyei G., Sato T., Geier J., Sawicki K.T, & Ardehali H. (2021). Augmenter of liver regeneration regulates cellular iron homeostasis by modulating mitochondrial transport of ATP-binding cassette B8. eLife, 10, e65158.

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Isoflurane-Induced Neuronal Cell Death in Co-Cultures

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Primary neuronal and mixed neuronal-astrocyte co-cultures at neuronal DIV 7 were exposed for 1 hr to either 2% isoflurane (assessed with a Datex 245 Airway Gas Monitor, Datex Corp., North Clearwater, Fl, USA) in pre-mixed carrier gas (5% C0₂, 21% O₂, balance N₂), or to carrier gas alone. Gas flow was maintained at a rate of 2 L/min in a sealed, humidified incubator at 37°C. Neurons were assessed for cell death 24 hrs following isoflurane exposure. In additional experiments, isoflurane-induced neuronal cell death was assessed in primary neuronal cultures following application of conditioned medium from neuronal-astrocyte co-cultures, in co-cultures following astrocyte-targeted knockdown of p75^NTR, and in co-cultures with and without the astrocyte-glutamate transporter inhibitor dihydrokainic acid (Tocris Bioscience, Bristol, UK). Finally, neuronal cell death was assessed in neuronal, astrocyte and neuronal-astrocyte co-cultures 24 hrs following application of recombinant proBDNF (1–100 pg/ml, R+D Systems, Minneapolis, MN, USA) plus protease inhibitor (diluted to 1X, G-Biosciences, St. Louis, MO), with and without the p75^NTR inhibitor trans-activating transcriptional activator peptide 5 (TAT-Pep5, 10 μM, EMD Millipore, Billerica, MA).

Stary C.M., Sun X, & Giffard R.G. (2015). Astrocytes Protect Against Isoflurane Neurotoxicity by Buffering pro Brain-Derived Neurotrophic Factor. Anesthesiology, 123(4), 810-819.

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Western Blot Reagents and Antibodies

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Western blotting reagents, including buffers and gels, were obtained from Bio-Rad Laboratories (Hercules, CA, USA). NP-40 lysis buffer and 4x Laemmle sample buffer were purchased from Alfa Aesar (Haverhill, MA, USA). ThioS, bovine serum albumin (BSA), and donkey serum were purchased from Sigma-Aldrich (St. Louis, MO, USA). Non-fat dry milk was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Protease inhibitor and FemtoLUCENT™ PLUS-HRP were obtained from G-Biosciences (St. Louis, MO, USA). Pierce ECL WB substrate was purchased from Thermo-Fisher Scientific (Waltham, MA, USA). All other chemicals were purchased from VWR (Radnor, PA, USA) or Fisher Scientific (Hampton, NH, USA). Antibodies used for Western blot (WB) and IHC are summarized in Table 1.

Abdallah I.M., Al-Shami K.M., Alkhalifa A.E., Al-Ghraiybah N.F., Guillaume C, & Kaddoumi A. (2023). Comparison of Oleocanthal-Low EVOO and Oleocanthal against Amyloid-β and Related Pathology in a Mouse Model of Alzheimer’s Disease. Molecules, 28(3), 1249.

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Western Blot Protein Quantification Protocol

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Protein levels were analyzed using western blot. Briefly, cells were harvested and washed with 1× phosphate buffered saline and lysed in a lysis buffer (RIPA buffer), Na-vanadate (1 mM), β-glycerol phosphate (50 mM), protease inhibitor (cat#786-108, G-biosciences, St. Louis, MO), EDTA (5 mM), β-mercaptoethanol (cat#41300000-1, 142 mM, BioWORLD, Dublin, OH). The cell lysate was mixed with 5× sample buffer and the mixture was boiled at 100°C for 10 min. Subsequently, the samples were loaded on 12% and 15% polyacrylamide gels. Proteins were transferred to membranes using Mini Trans-Blot Cell and Criterion Blotter (Bio-Rad, Hercules, CA) and the membranes were blocked in 1% BSA (bovine serum albumin, cat#160069, MP Biomedicals, Santa Ana, CA) dissolved in Tris-buffered saline containing 0.1% Tween 20 (TBST). The membranes were probed by indicated primary antibodies and incubated on a rotator at 4°C overnight. Then, the membranes were washed for 5 minutes in TBST and treated with anti-mouse or -rabbit secondary antibodies for 1 hour at room temperature. After washing three times for 10 minutes each with TBST, proteins were detected using a chemiluminescent substrate (EZ west Lumi Plus, ATTO, Tokyo, Japan) and visualized on a chemiluminescence Imaging system (Luminograph II, ATTO).

Kim D.Y., Nam J., Chung J.S., Kim S.W, & Shin H.J. (2021). Role of Roflumilast Combined with ESHAP Chemotherapy in Relapsed/Refractory Patients with Diffuse Large B-Cell Lymphoma. Cancer Research and Treatment : Official Journal of Korean Cancer Association, 54(1), 301-313.

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