Protease inhibitor

Manufactured by Thermo Fisher Scientific

Sourced in United States, China, United Kingdom, Germany, France, Japan, Switzerland

Protease inhibitors are a class of chemical compounds used in laboratories to prevent the degradation of proteins by proteolytic enzymes. They function by binding to and inhibiting the catalytic activity of proteases, thereby protecting the integrity of protein samples during various analytical and experimental procedures.

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Lab products found in correlation

1 135 protocols using protease inhibitor

Immunoprecipitation of Protein Complexes

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The indicated plasmids were transfected into SMMC-7721 cells that had been treated with or without the proteasome inhibitor MG132. The cells were lysed in RIPA buffer (Beyotime Biotechnology) containing protease inhibitors and RNase Inhibitor (Life Technologies) and centrifuged at 16,400×g for 15 min. The supernatants were incubated with anti-FLAG or anti-HA Protein G Dynabeads (Life Technologies) overnight at 4 °C with gentle rotation. The beads were washed thrice with NT2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Nonidet P-40, 1 mM MgCl₂) containing protease inhibitors (Thermo Fisher Scientific Inc.) and RNase Inhibitor (Thermo Fisher Scientific Inc.) and twice with PBS containing protease inhibitors and RNase Inhibitor (Thermo Fisher Scientific Inc.). After washing, the proteins were eluted by competition with FLAG or HA peptides (Thermo Fisher Scientific Inc.). The immuno-complexes were analysed by SDS/PAGE and immunoblotting with anti-Flag, anti-HA, anti-IGF2BP1, anti-IGF2BP3 or anti-PRMT5 antibody.

Li Z., Zhang J., Liu X., Li S., Wang Q., Di C.h.e.n., Hu Z., Yu T., Ding J., Li J., Yao M., Fan J., Huang S., Gao Q., Zhao Y, & He X. (2018). The LINC01138 drives malignancies via activating arginine methyltransferase 5 in hepatocellular carcinoma. Nature Communications, 9, 1572.

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Subcellular Protein Fractionation in 143B Cells

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Approximately 2 × 10⁶ 143B cells were harvested with trypsin-EDTA solution (Thermo Fisher Scientific, 25200072) and washed with PBS. Next, 200 μL ice-cold cytoplasmic extraction reagent I (Thermo Fisher Scientific, 78833) with protease inhibitor (Thermo Fisher Scientific, 78430) was added to the dried cell pellets. The tube was vortexed at the highest setting for 15 s and incubated on ice for 10 min. After adding 11 μL ice-cold cytoplasmic extraction reagent II (Thermo Fisher Scientific, 78833), the tube was vortexed at the highest setting for a further 5 s and incubated on ice for 1 min. After centrifugation at 13000 x g for 5 min, the supernatant (cytoplasmic extract) was transferred to a clean pre-chilled tube. The insoluble fraction was then suspended in 100 μL nuclear extraction reagent (Thermo Fisher Scientific, 78833) and protease inhibitor. The cell pellets were then left on ice and vortexed for 15 s every 10 min for a total of 40 min. After centrifugation at 13000 x g, for 10 min, the supernatant (nuclear extract) was transferred to a new tube. Cytoplasmic and nuclear proteins were analyzed using western blotting.

Niu J., Yan T., Guo W., Wang W., Ren T., Huang Y., Zhao Z., Yu Y., Chen C., Huang Q., Lou J, & Guo L. (2022). The COPS3-FOXO3 positive feedback loop regulates autophagy to promote cisplatin resistance in osteosarcoma. Autophagy, 19(6), 1693-1710.

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Rapid Brain Extraction and Fractionation

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Rats were briefly exposed to isoflurane gas until effect and rapidly decapitated. The whole brain was extracted and immediately frozen in powdered dry ice and stored at −80°C until processing. The brain was sliced into 1mm coronal sections using the brain matrix and the NAc and the mPFC were dissected using the 1mm biopsy punches. A rat brain stereotaxic atlas (Paxinos and Watson 1998 ) was used to identify regions of interest. The tissue punches from each brain region were homogenized separately in extraction buffer (150mM NaCl, 50mM Tris-Base pH 8.0)+EDTA+protease inhibitors (ThermoScientific Protease cocktail) or in experiment 2 tissue punches were placed in 1ml of sucrose harvest buffer (0.32M sucrose, 2mM EDTA, 50mM Tris-HCl pH 7.4)+protease inhibitor (ThermoScientific Protease cocktail) and homogenized. The homogenized samples were stored in the sucrose harvest buffer at −80°C until the subcellular fractionation was completed.

Garcia E.J., Arndt D.L, & Cain M.E. (2019). Dynamic Interactions of Ceftriaxone and Environmental Variables Suppress Amphetamine Seeking. Brain research, 1712, 63-72.

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Protein Extraction from Spleen and Thymus

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The spleen and thymus tissues each were washed with 1X phosphate buffered saline (PBS) (Welgene, Daegu, Republic of Korea) in 1X protease inhibitor (Thermo Fisher Scientific, Rockford, IL, USA) 2 times for 5 min. While on dry ice, each tissue was minced using a scalpel, and the pieces were transferred to 1 mL of 1X RIPA buffer (Thermo Fisher Scientific) containing 1X protease inhibitor (Thermo Fisher Scientific) and 1X phosphatase inhibitor (Roche, PhosSTOP Easy pack, Mannheim, Germany). Then, each sample was ultrasonicated. Next, the supernatant was obtained by centrifugation at 4^°C and 8,000 g for 10 minutes. Bicinchoninic acid (BCA) quantification was performed with the Micro BCA Protein Assay Kit (Thermo Fisher Scientific).

Lee Y.Y., Seo H.W., Kyung J.S., Hyun S.H., Han B.C., Park S., So S.H., Lee S.H, & Yi E.C. (2019). Proteomic studies of putative molecular signatures for biological effects by Korean Red Ginseng. Journal of Ginseng Research, 43(4), 666-675.

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Gastric Cytokine Profiling in Mice

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Gastric tissue from male mice was homogenized in liquid nitrogen with a disposable pestle (Sigma-Aldrich, St. Louis, MO). One hundred fifty microliters of lysis buffer, 500 µL of RIPA buffer with protease inhibitor (Thermo Fisher Scientific, Waltham, MA), 5-µL protease inhibitor, and 5-µL 0.5 M EDTA were added. Samples were placed in a rotating mixer at 4°C for 1 hour. Supernatant was collected following centrifugation at 10,000 × g for 10 minutes at 4°C. Protein concentration was measured using a BCA kit (Thermo Fisher Scientific, Waltham, MA) and adjusted to 1 mg/mL. Thirty-two-plex gastric tissue cytokine array was performed to quantify eotaxin, G-CSF, GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17A, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNFα, and VEGF-A (Eve Technologies, Calgary, Alberta, Canada).

Lunger C., Shen Z., Holcombe H., Mannion A.J., Dzink-Fox J., Kurnick S., Feng Y., Muthupalani S., Carrasco S.E., Wilson K.T., Peek R.M., Piazuelo M.B., Morgan D.R., Armijo A.L., Mammoliti M., Wang T.C, & Fox J.G. (2023). Gastric coinfection with thiopeptide-positive Cutibacterium acnes decreases FOXM1 and pro-inflammatory biomarker expression in a murine model of Helicobacter pylori-induced gastric cancer. Microbiology Spectrum, 12(1), e03450-23.

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Protein-Peptide Binding Assay

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The recombinant mouse IL‐6 receptor subunit alpha protein with the Fc region of mouse IgG2a (Fc‐IL‐6Rα, abcam) and mouse IL‐21 receptor protein with the Fc region of human IgG1 (IL‐21R‐Fc, Sino Biological) were commercially purchased. Fc‐IL‐6Rα (0.5 µg) or IL‐21R‐Fc (0.5 µg) plus salivaricin A2 (5 µg), salivaricin B (5 µg), negative control peptides A2 (5 µg) or negative control peptides B (5 µg) were incubated in 1 mL of binding buffer (20 mM Tris‐HCl (pH 7.6), 200 mM NaCl, 1 mM EDTA, 2 × protease inhibitor (Thermo Scientific)) at 4 °C for 2 h on a rotation mixer. Subsequently, 10 µL of Protein A Agarose (Thermo Scientific) was added to the reaction system and incubated for an additional 2 h. After the incubation, a buffer containing 20 mM Tris‐HCl (pH 7.6), 200 mM NaCl, 1 mM EDTA, 0.6% Nonidet P‐40 (v/v), and 2 × protease inhibitor (Thermo Scientific) was used to wash the agarose 4 times, and a buffer containing 2% formic acid plus 50% acetonitrile was used to elute the agarose. Then, the samples were re‐suspended with 50 µL PBS, and detected the targeted proteins or peptides by LTQ‐Orbitrap Velos Mass Spectrometer, Q‐Exactive.

Li J., Jin J., Li S., Zhong Y., Jin Y., Zhang X., Xia B., Zhu Y., Guo R., Sun X., Guo J., Hu F., Xiao W., Huang F., Ye H., Li R., Zhou Y., Xiang X., Yao H., Yan Q., Su L., Wu L., Luo T., Liu Y., Guo X., Qin J., Qi H., He J., Wang J, & Li Z. (2022). Tonsillar Microbiome‐Derived Lantibiotics Induce Structural Changes of IL‐6 and IL‐21 Receptors and Modulate Host Immunity. Advanced Science, 9(30), 2202706.

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Platelet Membrane Cloaked Nanoparticles

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Bovine blood (Quad Five) was centrifuged at 200g for 25 min. The platelet rich plasma was extracted and centrifuged at 250g for 25 min. The blood cells were pelleted and removed, while the plasma was purified by adding DPBS containing ethylenediaminetetraacetic acid (EDTA) and prostaglandin E1 (Sigma) to prevent platelet activation. Then, the platelets were pelleted by centrifugation at 800g for 25 min and suspended in DPBS containing EDTA and protease inhibitors (Thermo Fisher). Platelet membrane vesicles were derived by performing 3–4 freeze–thaw cycles on the platelet suspension and collected by centrifugation at 4500g for 5 min. The membrane vesicles were washed with DPBS containing protease inhibitors and sonicated using an ultrasonic bath (130W, Fisher Scientific) for 5 min. To cloak platelet membrane onto the nanoparticles, 10 mg of oxygen-releasing nanoparticles were mixed with the membrane vesicles derived from 2 × 10¹⁰ platelets and sonicated for 5 min. The size and surface zeta potential of the nanoparticles before and after cloaking were measured by dynamic light scattering (Malvern ZEN3600) where the nanoparticles were suspended in water. The morphology was examined using transmission electron microscopy (TEM, FEI Tecnai G2 Spirit) where the dry nanoparticles were used.

Guan Y., Niu H., Wen J., Dang Y., Zayed M, & Guan J. (2022). Rescuing Cardiac Cells and Improving Cardiac Function by Targeted Delivery of Oxygen-Releasing Nanoparticles after or Even before Acute Myocardial Infarction. ACS nano, 16(11), 19551-19566.

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Protein Extraction and Immunoblotting Protocol

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Frozen skin tissue samples were homogenized in liquid nitrogen using a mortar and pestle. Protein lysate was extracted from tissues using 1x RIPA lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, and 1% sodium deoxycholate) plus 1x protease inhibitors (Fisher Scientific; Cat # 88666), and from cells using 1x lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Tween-20) plus protease inhibitors and to determine the protein levels, the immunoblotting protocol was followed as previously described by us (Gaddameedhi et al., 2011 (link); Gaddameedhi et al., 2015 (link)). The following antibodies were used: GAPDH (Santa Cruz Biotechnology; Cat # sc-25778), BMAL1 (Bethyl Laboratories; Cat # A302–616A), β-Tubulin and pH2A.X (Cell Signaling Technology; Cat #s 86298T and 9718S, respectively). The appropriate HRP-conjugated secondary antibody was used for detection with chemiluminescence (Clarity Western ECL, Bio-Rad, and/or SuperSignal West Femto, Thermo Fisher Scientific) using a Bio-Rad ChemiDoc XRS+ imager.

Kaplan D., Ferrari I., Bergami P.L., Mahler E., Levitus G., Chiale P., Hoebeke J., Van Regenmortel M.H, & Levin M.J. (1997). Antibodies to ribosomal P proteins of Trypanosoma cruzi in Chagas disease possess functional autoreactivity with heart tissue and differ from anti-P autoantibodies in lupus. Proceedings of the National Academy of Sciences of the United States of America, 94(19), 10301-10306.

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Isolation of Mouse Brain Microvessels

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Mouse brain microvessels were isolated as previously described (Park et al., 2013 (link); Andras et al., 2017 (link)). Briefly, brains were homogenized in cold isolation buffer (102 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 15 mM HEPES, 25 mM NaHCO₃, 10 mM glucose, 1 mM Na pyruvate) with freshly added protease inhibitors (Thermo Fisher Scientific) and filtered through a 300 μm nitrocellulose mesh filter (EMDMilipore). Then, 26% dextran (MW, 150 kDa) in isolation buffer was added to the filtered brain homogenate, mixed, and centrifuged at 5,800 × g for 20 min at 4°C. The supernatant was removed, pellets were resuspended in isolation buffer, and filtered through a 120 μm nitrocellulose mesh filter (EMDMilipore). Filtered homogenates were then re-pelleted by centrifugation (1,500 × g, 10 min, 4°C) and re-resuspended either in 200 μL of radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) supplemented with protease inhibitors for immunoblotting, or in PBS for RNA sequencing.

Leda A.R., Bertrand L., Andras I.E., El-Hage N., Nair M, & Toborek M. (2019). Selective Disruption of the Blood–Brain Barrier by Zika Virus. Frontiers in Microbiology, 10, 2158.

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HEK293T Cell Lysis and Protein Extraction

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HEK293T cell pellets were suspended in lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM DTT, 0.8% NP-40, 5% glycerol, and 1x protease inhibitors (Thermo)) at 2× pellet volume. The samples were sonicated on ice for 30 s at 20% amplitude with a 2 second pulse ON and 3 s pulse OFF. Cell debris was removed by centrifugation at 20,000 × g for 20 min at 4 °C. Protein concentration was determined by BCA (Pierce) and diluted down to 5 mg/mL with pulldown buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM DTT, 0.4% NP-40, 1× protease inhibitors (Thermo Fisher Scientific)).

Schröder M., Renatus M., Liang X., Meili F., Zoller T., Ferrand S., Gauter F., Li X., Sigoillot F., Gleim S., Stachyra T.M., Thomas J.R., Begue D., Khoshouei M., Lefeuvre P., Andraos-Rey R., Chung B., Ma R., Pinch B., Hofmann A., Schirle M., Schmiedeberg N., Imbach P., Gorses D., Calkins K., Bauer-Probst B., Maschlej M., Niederst M., Maher R., Henault M., Alford J., Ahrne E., Tordella L., Hollingworth G., Thomä N.H., Vulpetti A., Radimerski T., Holzer P., Carbonneau S, & Thoma C.R. (2024). DCAF1-based PROTACs with activity against clinically validated targets overcoming intrinsic- and acquired-degrader resistance. Nature Communications, 15, 275.

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