Protease inhibitor tablet

Manufactured by Thermo Fisher Scientific

Sourced in United States, China

Protease inhibitor tablets are used to inhibit the activity of proteases, which are enzymes that break down proteins. They serve as a tool for researchers to preserve the integrity of protein samples during various laboratory procedures.

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Protease inhibitors (1135 citations) Pierce Protease Inhibitor Tablets (54 citations) Pierce Protease and Phosphatase Inhibitor Mini Tablets (45 citations) Pierce Protease Inhibitor Mini Tablets (42 citations) Protease and phosphatase inhibitor mini tablets (21 citations) Protease Inhibitor Mini Tablets (18 citations) Protease inhibitor cocktail tablets (16 citations) Pierce protease and phosphatase inhibitor tablets (10 citations) Protease and phosphatase inhibitor tablets (8 citations) Protease and phosphatase inhibitor cocktail tablets (7 citations)

59 protocols using protease inhibitor tablet

MYOD mRNA and Protein Stability Assays

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To measure MYOD mRNA half-life at day 0 (following transfection of 54-1 cells with siControl #1 or siUPF1 #1), actinomycin D (ActD, Sigma, 2.5μg/mL) was added to cells to inhibit transcription. After 0, 0.5, 1, 2, 4, 8 and12 hours of ActD treatment, cells were collected with TRIzol (Invitrogen), RNA was extracted, and qPCR was carried out with primers specific to mature MYOD mRNA. To inhibit the 26S proteasome, MG132 (10μM) was added to MB135-Tet-UPF1^WT cells 12 hours post-Dox induction. After 8 hours of MG132 treatment, identical numbers of cells were collected in RIPA buffer (Cell Signaling) supplemented with protease inhibitor tablets (Fisher Scientific), protein was extracted, and immunoblots for MYOD were performed. To measure MYOD protein levels in MB135-Tet-UPF1^WT or MB135-Tet-UPF1^{S124A/N138A/T139A} cells that were or were not treated with Dox to induce transgenic UPF1 expression, cycloheximide (CHX, 100μg/mL) was added to cells 12 hours post-Dox induction to inhibit translation. After 0, 2, 4, 6 and 8 hours of CHX treatment, identical numbers of cells were collected in RIPA buffer (Cell Signaling) supplemented with protease inhibitor tablets (Fisher Scientific), protein was extracted, and immunoblots for MYOD were performed.

Feng Q., Jagannathan S, & Bradley R.K. (2017). The RNA surveillance factor UPF1 represses myogenesis via its E3 ubiquitin ligase activity. Molecular cell, 67(2), 239-251.e6.

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Biomechanical Analysis of Intervertebral Disc Degeneration

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Axial biomechanics are reflective of physiological loading and were used to evaluate NP pressurization, AF tension, and IVD laxity. Creep is known to be sensitive to alterations in short and long-time viscoelastic behaviors. Axial and creep testing used an ElectroForce 3200 instrument (TA Instruments, New Castle, DE). Samples were hydrated in PBS with protease inhibitor tablets (ThermoScientific, Rockford, IL). Motion segments underwent 20 cycles ±8N of tension-compression at 1Hz followed by 60 minutes of compressive creep at −8N and 30 minutes of unloaded rehydration. Compressive stiffness, tensile stiffness, axial range of motion, and axial hysteresis were calculated from the 20th cycle in MATLAB (Figure 3B). Three independent reviewers manually calculated neutral zone (NZ) length and stiffness. Samples with undetectable NZ were excluded from NZ stiffness analysis. Creep was analyzed with MATLAB code measuring total displacement and applying a 5-parameter viscoelastic solid model to calculate elastic response (S_e), fast response (τ₁ and S₁), and slow response (τ₂ and S₂) time constant and stiffness parameters, respectively (39 (link)) (Figure 3C).

Mosley G.E., Hoy R.C., Nasser P., Kaseta T., Lai A., Evashwick-Rogler T.W., Lee M, & Iatridis J.C. (2019). Sex differences in rat intervertebral disc structure and function following annular puncture injury. Spine, 44(18), 1257-1269.

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Transporters and Membrane Protein Analysis

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Phenolsulfonphthalein (PSP)
was obtained from Macklin (Shanghai, China). Estradiol-17β-glucuronide
(E17βG), estrone-3-sulfate (E3S), and taurocholate (TCA) were
purchased from Sigma-Aldrich (St. Louis, MO). 4-(4-(Dimethylamino)styryl)-N-methylpyridinium (ASP⁺) and penciclovir (PCV)
were from US Everbright (Suzhou, China) and Aladdin (Shanghai, China),
respectively. Dulbecco’s modified Eagle’s medium (DMEM),
PSP-free DMEM, fetal bovine serum (FBS), trypsin, Lipofectamine 2000,
sulfo-N-hydroxysuccinimide-SS-biotin,
protease inhibitor tablets, and streptavidin-agarose resin were purchased
from Thermo Fisher Scientific (Waltham, MA). Penicillin-streptomycin
was from Hyclone (Logan, UT). The bicinchoninic acid (BCA) protein
assay kit was purchased from Takara (Kyoto, Japan). The first antibodies
for detecting 6-His tag and Na⁺/K⁺-ATPase were
from Proteintech (Rosemont, IL) and Abcam (Boston, MA), respectively.
Their secondary antibodies were from Proteintech (Rosemont, IL).

Wang Z., Li Y., Peng T., Su Y., Luo X., Han W., Zhang H., Ruan J, & Gui C. (2021). Human Organic Anion Transporting Polypeptides 1B1, 1B3, and 2B1 Are Involved in the Hepatic Uptake of Phenolsulfonphthalein. ACS Omega, 6(51), 35844-35851.

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Immunoprecipitation of GFP-Tagged Proteins

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Nicotiana benthamiana leaves expressing the appropriate constructs were collected at 2 d postinfection. The cells were homogenized in liquid nitrogen and suspended in 500-μL protein extraction buffer (10% (v/v) glycerol; 25-mM Tris–HCl (pH 7.5); 1-mM EDTA; 150-mM NaCl; 10-mM DTT; 0.2% Nonidet P-40; 1-mM phenylmethylsulfonyl fluoride (PMSF); and protease inhibitor tablets (A32955; Thermo Fisher Scientific, Waltham, MA, USA)) followed by extraction on ice for 30 min and centrifugation for 20 min at 12,000 R/min at 4°C. Twenty microliters of prewashed anti-GFP agarose beads (http://www.ktsm-life.com/, KTSM1301) were added to 400-μL protein extracts and incubated for 2 h at 4°C. Then, the beads were washed 4 times with wash buffer (10% (v/v) glycerol; 25-mM Tris–HCl (pH 7.5); 1-mM EDTA; 150-mM NaCl; 1-mM PMSF; protease inhibitor tablets (A32955; Thermo Fisher Scientific). The immunoprecipitates were eluted with 1 × SDS loading buffer. The samples were boiled at 95°C for 10 min before discarding the beads and subsequently stored at −80°C until western blot analysis.

Jing S., Sun X., Yu L., Wang E., Cheng Z., Liu H., Jiang P., Qin J., Begum S, & Song B. (2022). Transcription factor StABI5-like 1 binding to the FLOWERING LOCUS T homologs promotes early maturity in potato. Plant Physiology, 189(3), 1677-1693.

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Automated TMT Proteomics Workflow

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Tandem Mass Tag (TMTpro) isobaric reagents, BCA protein concentration kit, protease inhibitor tablets, Pierce C18 tips, and trypsin were purchased from ThermoFisher Scientific (Rockford, IL). StageTip Empore-C18 material was purchased from CDSanalytical (Oxford, PA). Sep-Pak cartridges (100 mg) were from Waters (Milford, MA). Lys-C protease was from Fujifilm Wako (Richmond, VA). Mass spectrometry grade water and organic solvents were from J.T. Baker (Center Valley, PA). The S. cerevisiae strains were from Horizon Scientific (Cambridge, UK). Synthetic complete (SC) media was from Sunrise Science (San Diego, CA). Sera-Mag Speed Beads (cat. nos. 45152105050350 and 65152105050350) were from GE Life Sciences (Marlborough, MA).
The OT-2 robotic liquid handler was purchased from opentrons (New York, NY) and included the magnetic module, the temperature module, and two GEN2 8-channel pipets: 20 and 300 μL. Labware used on the OT-2 included: 12-channel reservoirs (USA Scientific, 1061-8150), 96-well microplate (Bio-Rad, HSP9601), 2 mL deep 96-well microplates (USA Scientific, 1896-2000), and 8-tube strip PCR tubes (Bio-Rad, TLS0801). Also, 8-tube strip PCR tube caps (Bio-Rad, TLS0803) are required for 37 °C incubations. Proteolytic digests were performed on the Jitterbug Heated Microplate Shaker (Boekel Scientific, 130000).

Liu X., Gygi S.P, & Paulo J.A. (2021). A Semiautomated Paramagnetic Bead-Based Platform for Isobaric Tag Sample Preparation. Journal of the American Society for Mass Spectrometry, 32(6), 1519-1529.

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Quantitative Proteomics of Microbes

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Tandem Mass Tag (TMT) isobaric reagents, BCA Protein Concentration Kit, Protease inhibitor tablets, and trypsin were purchased from ThermoFisher Scientific (Rockford, IL). StageTip Empore-C18 material was purchased from CDS (Oxford, PA). Sep-Pak cartridges (50 mg) were from Waters (Milford, MA). LysC protease was from Fujifilm Wako (Richmond, VA). Mass spectrometry-grade water and organic solvents were from J.T. Baker (Center Valley, PA). The S. cerevisiae strain used was BY4716 from ThermoFisher Scientific (Waltham, MA). The S. pombe strain was NRRL Y-164 purchased from ATCC (Manassas, Virginia). The E. coli strain was purchased from Carolina Biological Supply Co. (Burlington, NC). Synthetic complete (SC), Edinburgh Minimal Media (EMM), yeast extracts with supplements (YES), and yeast-peptone-dextrose (YPD) media was from Sunrise Science (San Diego, CA). Luria-Bertani broth (LB) and Nutrient broth #1 (NB) was purchased from Millipore-Sigma (St. Louis, MO). Media formulations are available in the Supplemental Methods. Sera-MagSpeed Beads (cat. nos. 45152105050350 and 65152105050350) were from GE Life Sciences (Marlborough, MA).

Navarrete-Perea J., Gygi S.P, & Paulo J.A. (2020). Growth media selection alters the proteome profiles of three model microorganisms. Journal of proteomics, 231, 104006.

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Cycloheximide Protein Turnover Assay

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AML12 cells were incubated at 90% confluency in complete growth media containing the protein synthesis inhibitor cycloheximide (Enzo Life Sciences) (50 µM final concentration) or vehicle (DMSO). Cells were collected in RIPA buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% Igepal, 0.5% Na-Deoxycholate, 0.1% SDS) containing proteinase inhibitors (ThermoScientific Protease-Inhibitor tablets, 2 tablet/10 ml buffer) at 0, 2, 4, 6, 8, and 24 h after incubation. The lysate was sonicated, centrifuged for 10 min at 18,800 × g and the resulting supernatants analyzed by immunoblotting.

Lee Y.A., Noon L.A., Akat K.M., Ybanez M.D., Lee T.F., Berres M.L., Fujiwara N., Goossens N., Chou H.I., Parvin-Nejad F.P., Khambu B., Kramer E.G., Gordon R., Pfleger C., Germain D., John G.R., Campbell K.N., Yue Z., Yin X.M., Cuervo A.M., Czaja M.J., Fiel M.I., Hoshida Y, & Friedman S.L. (2018). Autophagy is a gatekeeper of hepatic differentiation and carcinogenesis by controlling the degradation of Yap. Nature Communications, 9, 4962.

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Melatonin's Impact on BM-MSCs

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BM-MSCs were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Tissue culture polystyrene plates and flasks were purchased from Costar (Tewksbury, MA, USA). Protease inhibitor tablets, fetal bovine serum (FBS), alpha minimum essential medium (α-MEM), Dulbecco’s modified Eagle medium (DMEM), horseradish peroxidase-conjugated secondary antibodies, SuperSignal West Pico Substrate, and CL-XPosure Film were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Penicillin, streptomycin, 4′,6-diamidino-2-phenylindole (DAPI), and TRIzol^® reagent were purchased from Invitrogen (Carlsbad, CA, USA). Melatonin, luzindole, sirtinol, H₂O₂, propidium iodide (PI), RNase A, dexamethasone, L-ascorbic acid, β-glycerophosphate, and fluorescein diacetate (FDA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Stock solution of Melatonin was prepared by dissolving in ethanol to the concentration of 200 mM, and diluting in α-MEM to different concentrations as specified in individual experiments. Primary antibodies against p16^INK4α, SIRT1, p38, p38 (phospho T180 + Y182), and α-tubulin were purchased from Abcam (Cambridge, MA, USA).

Zhou L., Chen X., Liu T., Gong Y., Chen S., Pan G., Cui W., Luo Z.P., Pei M., Yang H, & He F. (2015). Melatonin reverses H2O2-induced premature senescence in mesenchymal stem cells via the SIRT1-dependent pathway. Journal of pineal research, 59(2), 190-205.

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Tandem Mass Tag Proteomics

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Paulo J.A., Navarrete-Perea J, & Gygi S.P. (2019). Multiplexed Proteome Profiling of Carbon Source Perturbations in Two Yeast Species with SL-SP3-TMT. Journal of proteomics, 210, 103531.

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Compound 22 Cytotoxicity Assay in A375 Cells

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A375 cells were plated in 100 mm dishes and cultured overnight at 37 °C at a density of 1.4 × 10⁶, 0.7 × 10⁶ and 0.4 × 10⁶ cells per dish for 24, 48 and 72 h respectively Next day, cells were treated with 100 μM of compound 22 for 24, 48 and 72 h and then trypsinized, washed twice with ice-cold PBS and pellets were collected after centrifugation at 2000 rpm for 3 min at 4 °C. Cell pellets were then lysed in lysis buffer (10 mM HEPES at pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl₂, 0.2% NP40) and supplemented with Protease Inhibitor Tablets (Thermo Scientific, Waltham, MA, USA). Then, they were left on ice while periodically vortexed over a 30 min period and sonicated (three cycles at 10 amplitudes for 20 s on ice) to disrupt cellular membranes. Cell lysates were centrifuged at full speed (15,000 rpm) for 10 min at 4 °C and supernatants were transferred in new tubes. Protein content was determined by utilizing the BCA protein assay kit (Thermo Scientific, Waltham, MA, USA), according to the manufacturer’s protocols. Protein extracts were stored at −20 °C until usage.

Kyriakou S., Mitsiogianni M., Mantso T., Cheung W., Todryk S., Veuger S., Pappa A., Tetard D, & Panayiotidis M.I. (2019). Anticancer activity of a novel methylated analogue of L-mimosine against an in vitro model of human malignant melanoma. Investigational New Drugs, 38(3), 621-633.

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