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Protease inhibitor cocktail 3

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Protease inhibitor cocktail III is a laboratory reagent used to inhibit the activity of proteases, which are enzymes that break down proteins. It is a mixture of several different protease inhibitors that can be effective against a broad range of proteases found in biological samples. The product is used in various research and experimental applications where it is necessary to preserve the integrity of proteins during sample preparation or analysis.

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Lab products found in correlation

Anti topk antibody, by BD (3 mentions) Anti foxm1 antibody, by Santa Cruz Biotechnology (3 mentions) Ip lysis buffer, by Thermo Fisher Scientific (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Anti β actin, by Merck Group (2 mentions) Sds page gel, by Bio-Rad (2 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions) Enhanced chemiluminescence detection reagent, by GE Healthcare (1 mentions) Anti ha, by Roche (1 mentions) Cl 1000 crosslinker, by Analytik Jena (1 mentions) Rnasin, by Promega (1 mentions) Complete edta free protease inhibitor cocktail, by Roche (1 mentions) Pegmal, by Merck Group (1 mentions) Halt phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Gtx 109000, by GeneTex (1 mentions) Phosphatase inhibitor cocktail 3, by Merck Group (1 mentions) Synergy h1, by Agilent Technologies (1 mentions) Antibiotic antimycotic mix, by Thermo Fisher Scientific (1 mentions) A68930, by Bio-Techne (1 mentions) Sch39166, by Bio-Techne (1 mentions)

Close spelling variants of this product

Protease inhibitor cocktail (9861 citations) Protease inhibitors (2755 citations) Proteases inhibitor cocktail (23 citations) Cocktail inhibitor (6 citations) Protease cocktails (3 citations) Protease inhibitor cocktails 2 and 3 (3 citations) Protease Inhibitor III (2 citations) Protease inhibitor cocktails set III (2 citations) Protease inhibitor and phosphatase inhibitor (2,3) cocktails (1 citations)

28 protocols using protease inhibitor cocktail 3

1

Western Blot Analysis of Signaling Proteins

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Western blot analysis was performed by normalization to β-actin or the baseline expression level. Cells were lysed with IP lysis buffer (Thermo Scientific, Waltham, MA) containing protease inhibitor cocktail III (Millipore, Billerica, MA). The proteins were separated by electrophoresis using 4-20% or 7.5% SDS-PAGE gel, and transferred onto nitrocellulose membrane. The membranes were incubated with the first antibody, respectively: anti-TOPK antibody (BD Biosciences, San Jose, CA), anti-FOXM1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-HA (Roche), or anti-β-actin (Sigma-Aldrich). Finally, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody and protein bands were visualized by enhanced chemiluminescence detection reagents (GE Healthcare, Pittsburgh, PA). We generated mouse anti-MELK monoclonal antibodies using partial recombinant MELK protein (264-601 amino acids of MELK) as an immunogen by the methods as described previously [31 (link)].
Kato T., Inoue H., Imoto S., Tamada Y., Miyamoto T., Matsuo Y., Nakamura Y, & Park J.H. (2016). Oncogenic roles of TOPK and MELK, and effective growth suppression by small molecular inhibitors in kidney cancer cells. Oncotarget, 7(14), 17652-17664.
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2

Immunoprecipitation of Myc-SYNJ2Arr-Mmito

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HEK293T cells were grown in 6 well plates and transfected with 3 μg/well Myc-SYNJ2Arr-Mmito or its VQL/AAA mutant. UV irradiation was performed by washing the cells with PBS and placing the plate on ice in a CL-1000 crosslinker (UVP) and exposing the plate to 400 mJ/cm2 254 nm UV light. After irradiation, cells were harvested in Lysis Buffer (1% Triton, 20mM Tris pH 7.4, 200mM NaCl, RNAsin (1:100 Promega), Protease inhibitor cocktail III (Millipore) and 200 μM PMSF) and cleared by centrifugation at 12000 g for 1 min. The supernatant was incubated with 3 μl anti-myc antibody (mouse 9E10, Novus) /mL lysate for 1h at 4°C. ProteinA sepharose beads were blocked with 3% BSA in lysis buffer for 30min, washed with PBS and added to the lysate. After 30 min incubation at 4°C beads were collected by centrifugation at 2000g for 30sec and washed three times with lysis buffer. Samples were eluted by addition of Laemmli Buffer and boiling at 95°C for 3 min prior to analysis by gel electrophoresis and immunoblotting with Rabbit myc-tag antibody, 71D10 (Cell Signaling).
Harbauer A.B., Hees J.T., Wanderoy S., Segura I., Gibbs W., Cheng Y., Ordonez M., Cai Z., Cartoni R., Ashrafi G., Wang C., Perocchi F., He Z, & Schwarz T.L. (2022). Neuronal mitochondria transport Pink1 mRNA via Synaptojanin 2 to support local mitophagy. Neuron, 110(9), 1516-1531.e9.
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3

STRA6 Recombinant Protein Production

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STRA6 from zebrafish (Danio rerio) was cloned into
pIEX/Bac-1 plasmid modified to include a C-terminal decahistidine tag and
thrombin cleavage site and transfected together with baculovirus bacmid DNA
(Sapphire Baculovirus DNA and Transfection kit, Allele Biotech) into
Spodoptera frugiperda Sf9 cells for generation of
recombinant baculovirus. The recombinant virus was amplified to high titer
following standard procedures. Sf9 cells were infected at a density of 2
× 106 cells per ml with high titer virus at an approximate
multiplicity of infection (MOI) of 1, grown in shaker flasks for 72 hours at
27°C and harvested by centrifugation. Cell pellets were resuspended in
low salt buffer consisting of 10 mM HEPES pH 7.5, 10 mM KCl, 10 mM
MgCl2, 0.5 mM PMSF, EDTA-free complete protease inhibitor
cocktail (Roche), DNase, and RNase and lysed by gentle brane fractions were
isolated by ultracentrifugation, resuspended by homogenization and washed a
minimum of two times, until the supernatant following ultracentrifugation was
clear, in high salt buffer (10 mM HEPES pH 7.5, 10 mM KCl, 1 M NaCl, 10 mM
MgCl2, 0.5 mM PMSF, EDTA-free complete protease inhibitor tablet
DNase, RNase). Extensively washed membranes were then resuspended again by
homogenization in buffer containing 30 mM HEPES, pH 7.5, 200 mM NaCl, 0.5 mM
PMSF, protease inhibitor cocktail III (Millipore) and stored at
−80°C until use.
Chen Y., Clarke O.B., Kim J., Stowe S., Kim Y.K., Assur Z., Cavalier M., Godoy-Ruiz R., von Alpen D.C., Manzini C., Blaner W.S., Frank J., Quadro L., Weber D.J., Shapiro L., Hendrickson W.A, & Mancia F. (2016). Structure of the STRA6 receptor for retinol uptake. Science (New York, N.Y.), 353(6302), aad8266.
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4

Protein Redox State Analysis in Fly Lysates

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Inspired by 4 (link). Flies were lysed in lysis buffer (50 mM Tris pH7.5, 1% Triton, 300 mM NaCl, 5 mM EDTA, 1:1000 Protease Inhibitor Cocktail III–#539134, Millipore), and debris was removed by centrifugation at 16,200 ×g. Laemmli buffer and 50 mM DTT were added to supernatant and the sample was boiled at 95°C for 5 min. Or flies were lysed in lysis buffer containing 1 mg/ml methoxypolyethylene glycol maleimide 5000 (PEG-MAL; 63187, Sigma), followed by centrifugation, and Laemmli buffer was added to supernatant at 37°C for 30 min. Or flies were lysed in lysis buffer containing 10 mM tris(2-carboxyethyl)phosphine (TCEP; 51805–45-9, Sigma), followed by centrifugation. Supernatant was incubated at 37°C for 30 min, then Laemmli buffer together with 15 mM 4-Acetamido-4’-Maleimidylstilbene-2,2’-Disulfonic Acid, Disodium Salt (AMS; A485, Thermo Fisher) or 1 mg/ml PEG-MAL was added, and the sample was incubated at 37°C for 10 min. Alternatively, mitochondrial pellet isolated from flies was suspended in mitochondrial isolation buffer, and treated with 50 mM DTT or 15 mM AMS at 37°C for 10 min. Or mitochondrial fraction was treated with 10 mM TCEP at 37°C for 30 min, pelleted via centrifugation and resuspended again, then Laemmli buffer containing 15 mM AMS was added, and the sample was incubated at 37°C for 10 min.
Li L., Conradson D.M., Bharat V., Kim M.J., Hsieh C.H., Minhas P.S., Papakyrikos A.M., Durairaj A.S., Ludlam A., Andreasson K.I., Partridge L., Cianfrocco M.A, & Wang X. (2021). A Mitochondrial Membrane-Bridging Machinery Mediates Signal Transduction of Intramitochondrial Oxidation. Nature metabolism, 3(9), 1242-1258.
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5

Neutrophil Elastase Quantification in BALF

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Mice were euthanized with a lethal dose of sodium pentobarbital (Socumb 6G, 150 mg/kg). Lungs were inflated via tracheal cannulation with 1 ml ice-cold PBS with protease inhibitors (protease inhibitor cocktail III; Millipore), and the liquid was retracted to collect bronchoalveolar lavage fluid (BALF). A total of 3 ml BALF was collected from each mouse. One milliliter of each sample was centrifuged at 21,000 × g for 5 min at 4°C. The supernatant was drawn into a 1-ml syringe and filtered through a 0.22-μm filter to remove bacteria. The centrifugation and filtration steps were repeated, and a portion of the sample was plated on BHI agar to verify the removal of Y. pestis. Samples were removed from the biosafety level 3 laboratory in accordance with CDC and UNC biosafety guidelines. Dilutions of each BALF sample were made and analyzed for the presence of elastase using a DuoSet Mouse Neutrophil Elastase/ELA2 enzyme-linked immunosorbent assay (ELISA) kit (R&D systems) as specified by the manufacturer’s protocol.
Eichelberger K.R., Jones G.S, & Goldman W.E. (2019). Inhibition of Neutrophil Primary Granule Release during Yersinia pestis Pulmonary Infection. mBio, 10(6), e02759-19.
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6

Cell Lysis and Protein Extraction

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After the appropriate treatments, cell monolayers were rinsed twice with 2 ml of chilled PBS, lysed with 500 μl of ice-cold lysis buffer consisting of 50 mM Tris buffer pH 8.0, 0.5% (w/v) sodium deoxycholate, 0.1% (v/v) protease inhibitor cocktail III (Millipore, Watford, UK) and 1% (v/v) HALT™ Phosphatase Inhibitor Cocktail (1:100; Thermo Fisher Scientific, Paisley, UK). Cell lysates were scraped and clarified by centrifugation at 4 °C for 20 min at 14 000 × g. Supernatants were collected and stored at − 20 °C. Protein concentration was measured by the bicinchoninic acid (BCA) protein assay, based on the method of Stoscheck (1990 ) using a kit from Sigma Aldrich (Poole, UK). For Western blot analysis, cell monolayers were rinsed as above, then lysed with 500 μl of 0.1% (w/v) sodium dodecyl sulphate (SDS) in PBS (pre-heated to 100 °C), after which the suspension was heated at 100 °C for 5 min and then centrifuged at 100,000 g for 30 min, before supernatants were collected and stored at − 20 °C.
Almami I.S., Aldubayan M.A., Felemban S.G., Alyamani N., Howden R., Robinson A.J., Pearson T.D., Boocock D., Algarni A.S., Garner A.C., Griffin M., Bonner P.L, & Hargreaves A.J. (2020). Neurite outgrowth inhibitory levels of organophosphates induce tissue transglutaminase activity in differentiating N2a cells: evidence for covalent adduct formation. Archives of Toxicology, 94(11), 3861-3875.
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7

Quantitative Analysis of A2M and MMP-2

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Culture supernatants were cleared by centrifugation (400× g, 3 min) and combined with 6× Laemmli buffer plus 2% β-mercaptoethanol. Equal volumes of culture supernatants from every tested condition were analyzed in each experiment. Cells were lysed in 1% Triton X-100, 0.1% SDS, 40 mM Tris-HCl pH 7.6, 150 mM NaCl, and protease inhibitor cocktail III (EMD Millipore, Merck, Darmstadt, Germany) for 15 min at 4 °C. Lysates were cleared by centrifugation (15,700× g, 5 min at 4 °C) and combined with 2× Laemmli buffer plus 2% β-mercaptoethanol. Equal volumes of cell lysates from every tested condition in each experiment were analyzed for GAPDH levels and used as a loading control proxy for A2M and MMP-2 levels in culture supernatants. Non-boiled samples were run in SDS-polyacrylamide gels and analyzed by Western blot using mouse anti-human A2M antibody (1:1000, clone 2D9, cat. ab36995, Abcam, Cambridge, UK) and rabbit polyclonal antibodies against human MMP-2 (1:1000, cat. 4022, Cell Signaling Technology, Danvers, MA, USA) and GAPDH (1:2000, cat. 2275-PC-100, Trevigen, Gaithersburg, MD, USA). The original, unmodified, and uncropped images from all Western blots presented are shown in Supplementary Figure S1.
Lehmann G.L., Ginsberg M., Nolan D.J., Rodríguez C., Martínez-González J., Zeng S., Voigt A.P., Mullins R.F., Rafii S., Rodriguez-Boulan E, & Benedicto I. (2022). Retinal Pigment Epithelium-Secreted VEGF-A Induces Alpha-2-Macroglobulin Expression in Endothelial Cells. Cells, 11(19), 2975.
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8

Proteomic Analysis of Colorectal Cancer

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The cancer tissues and paracancer tissues of eight CRC patients from colon segment were ground into cell powder by liquid nitrogen. Then, the mixture was next crushed by the ultrasonic equipment (Scientz) with lysis buffer [8 M urea and 1% protease inhibitor cocktail III (Merck Millipore, 156535140)]. The preceding steps were performed on ice. After that, the crushing liquid was centrifuged at 12000 g for 10 min at 4°C, and the supernatant was collected to determine the protein concentration using the BCA kit (Beyotime P0011-1) as directed by the manufacturer.
Following that, trichloroacetic acid (TCA) was added to the ultrasonic lysate at a final concentration of 20%. After storage at 4°C for 2 h, the mixture was centrifuged at 4500 g for 5 min at 4°C. Then, the supernatant was removed, and the precipitation was washed with pre-cooled acetone 2-3 times. Then triethylammonium bicarbonate (TEAB) was added to the precipitation, bringing the final concentration to 200 mM. After centrifugation, trypsin was injected in a 1:50 ratio for incubation. After that, dithiothreitol (DTT) was employed to the mixture staying at 56°C for 30 min (final concentration = 5 mM). Finally, the mixture was added with Iodoacetamide (IAA) (final concentration = 11 mM) and sit at room temperature in the dark for 15 min.
Zhong Y., Zhang W., Yu H., Lin L., Gao X., He J., Li D., Chen Y., Zeng Z., Xu Y., Tang D, & Dai Y. (2022). Multi-platform-based characterization of ferroptosis in human colorectal cancer. iScience, 25(8), 104750.
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9

Western Blot Analysis of Protein Expression

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Either fresh cell pellets or frozen cell pellets thawed on ice were
resuspended in lysis buffer (50 mMTrisHCl pH 7.4,100 mM NaCl, 1% NP-40,0.1%
SDS, 0.5% sodium deoxycholate, 1:200 Protease Inhibitor Cocktail III (EMD
Millipore)).The suspensions were then sonicated for five cycles of 30 s
intervals at 4°C. Protein was quantified using the BCA assay
(Pierce). 30 μg of protein was heat denatured and run on
4%−12% NuPAGE Novex Bis-Tris protein gels (ThermoFisher) and
transferred to PVDF membranes. The membranes were then blocked in 5% milk in
TBS buffer for 1h at 25°C. Membranes were incubated in primary
antibody diluted in 5% milk TBS-tween overnight at 4°C. Primary
antibodies used were: GAPDH (Abcam ab8245; 1:10,000), α-TUB (Abcam
ab7291, 1:5000), ILF3 (Bethyl A303–651A; 1:10,000), LIN28B (Bethyl
A303–588A; 1:2,000), PRPF8 (Bethyl A303–921A; 1:10,000), PTBP1
(MBL RN011P; 1:5,000), SF3B4 (Bethyl A303–950A; 1:10,000), DDX3X
(Bethyl A300–474A; 1:1000), SLTM (Bethyl A302–834A; 1:1000),
LARP7 (Bethyl A303–723A; 1:1000), LARP4 (Bethyl A303–900A;
1:1000), DKC1 (Genetex GTX109000; 1:1000), RBFOX2 (Bethyl A300–864A;
1:1000), BUD13 (Bethyl A303–320A; 1:1000). Membranes were washed and
probed with a 1:10,000 dilution of secondary antibody prepared in 5% milk
TBS-tween for 1 hour at 25°C, washed, and developed with ECL or ECL+
(Pierce).
Nussbacher J.K, & Yeo G.W. (2018). Systematic Discovery of RNA Binding Proteins that Regulate MicroRNA Levels. Molecular cell, 69(6), 1005-1016.e7.
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10

Fluorescence-based Kinase Assay Protocol

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All assays were performed in 384-well, white, half-area plates (Corning, 3824) with a total volume of 40 μL. Fluorescence data were collected with a Bio-Tek Synergy H1 microplate reader with excitation at 360 nm and emission at 485 nm. Assay buffer consisted of 50 mM Tris-HCl (pH 7.5 at 22 °C), 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, 0.01% Brij-35-P, and 1 mM ATP. Lysis buffer consisted of 50 mM Tris-HCl (pH 7.5 at 22 °C), 150 mM NaCl, 30 mM NaF, 1% (v/v) Triton X-100, 2 mM EGTA, 1 mM DTT, 1% (v/v) protease inhibitor cocktail III (EMD Millipore, 539134), and 1% (v/v) phosphatase inhibitor cocktail III (Sigma, P0044). Detailed methods for probe synthesis as well as tissue lysis and analysis can be found in previously published protocols,22 (link),23 (link) brief descriptions of these procedures are given below.
Beck J.R., Cabral F., Rasineni K., Casey C.A., Harris E.N, & Stains C.I. (2019). A Panel of Protein Kinase Chemosensors Distinguishes Different Types of Fatty Liver Disease. Biochemistry, 58(37), 3911-3917.
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