Protease inhibitor cocktail 1x

Manufactured by Merck Group

Sourced in China

Protease inhibitor cocktail 1X is a laboratory reagent used to inhibit the activity of proteases, a class of enzymes that break down proteins. It is a ready-to-use solution that can be added to cell or tissue lysates to prevent protein degradation during sample preparation and analysis.

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Cocktail protease inhibitors/1X Protease inhibitor cocktail Proteases inhibitor cocktail Protease cocktails Cocktail inhibitor Protease inhibitors

5 protocols using protease inhibitor cocktail 1x

HDAC4 Protein Extraction and Quantification

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20–30 mg of frozen tissues were homogenated in 200 μl modified lysis buffer (25 mM Tris-HCl pH 7.6, 2 mM EDTA pH 8, 250 mM Sucrose, 0.1% TritonX-100, 5 mM DTT, 1 mM PMSF, 200 mM SOV4, 10 mM NaF, protease inhibitor cocktail 1X; Sigma-Aldrich). A standard RIPA buffer (25 mM Tris-HCl, 1% NP-40, 1% Na-deoxycholate, 150 mM NaCl, 1 mM PMSF, 1 mM NaF, 1 mM Na3VO4, protease inhibitor cocktail 1X; Sigma-Aldrich) was instead used for the extraction of total proteins from cell cultures. Protein samples were quantified by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and stored at −20 °C.
20 µg of lysate samples were separated on Mini-PROTEAN TGX Stain-Free Gels (4–20%, Bio-Rad). The gel was transferred to a nitrocellulose membrane (Bio-Rad) for 10 min using the Trans-Blot Turbo Transfer System (Bio-Rad) with Trans-Blot Turbo Mini Transfer Packs (Bio-Rad). The membrane was activated for 1 minute and then incubated with an antibody against HDAC4 (Cell Signaling, #2072) at a dilution of 1:500. Immune complexes were detected using the ChemiDoc Touch Imaging System (Bio-Rad)after incubation with Clarity Western ECL Substrate (Bio-Rad). HDAC4 protein expression was quantified and normalized to stain free gel loading using ImageLab software (version 5.2.1, Bio-Rad).

Di Pietro L., Baranzini M., Berardinelli M.G., Lattanzi W., Monforte M., Tasca G., Conte A., Logroscino G., Michetti F., Ricci E., Sabatelli M, & Bernardini C. (2017). Potential therapeutic targets for ALS: MIR206, MIR208b and MIR499 are modulated during disease progression in the skeletal muscle of patients. Scientific Reports, 7, 9538.

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Immunoprecipitation Workflow for Protein Analysis

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For immunoprecipitation, cells were lysed in ice-cold Co-IP buffer (50
mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 150 mM NaCl, and 0.5% NP40) in the
presence of 1 mM PMSF, protease inhibitor cocktail (1X), and phosphatase
inhibitor cocktail (Sigma) and then incubated on ice for 30 min. Lysates were
pre-cleared by incubating with Protein A Sepharose and rotating overnight. The
next day, the pre-cleared lysates were incubated with primary antibody for 3
hours by rotating at 4°C. Then the samples were incubated with Sepharose
Protein A/G beads for an additional 2 hours. Subsequently, the beads were
pelleted and washed three times in Co-IP buffer, and then resuspended in Laemmli
buffer containing beta-mercaptoethanol for immunoblot analysis.

Golas G.J., Jang S.J., Naik N.G., Alonso J.D., Papp B, & Toth Z. (2019). Comparative analysis of the viral interferon regulatory factors of KSHV for their requisite for virus production and inhibition of the type I interferon pathway. Virology, 541, 160-173.

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Size-Exclusion Chromatography of CFA Synthase

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Size-exclusion chromatography was performed using a Superdex 200 10/300 GL column (GE Healthcare) equilibrated in storage buffer. For tryptic cleavage, CFA synthase (2 nmol in 100 μL) was incubated with trypsin (3 μg/mL, Sigma) for 30 min at 22 °C and quenched with protease inhibitor cocktail (1X, Sigma) before loading onto the column.

Hari S.B., Grant R.A, & Sauer R.T. (2018). Structural and Functional Analysis of E. coli Cyclopropane Fatty Acid Synthase. Structure (London, England : 1993), 26(9), 1251-1258.e3.

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Western Blot Protocol for Protein Analysis

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MB cell lines were harvested and lysed by RIPA buffer (Beyotime, Nantong, China) supplied with protease inhibitor cocktail (1x) (Sigma-Aldrich). Protein concentrations were measured by Bradford assay. An equal amount of protein (40 μg) was loaded onto a 10% SDS-PAGE gel. Proteins in the gel were electro-transferred to PVDF (polyvinylidene difluoride) membranes, which were blocked with 5% BSA for 1 h. Membrane was incubated with diluted primary antibodies (1:1000) in PBST at 4°C for overnight. After washing with PBST for 3 times of 5 min each, membranes were incubated with secondary antibody at room temperature for 1 h. The proteins from membranes were detected by Hyperfilm-ECL kits (GE Healthcare Biosciences). β-actin was a loading control.

Ge J., Wang B., Zhao S, & Xu J. (2022). Inhibition of lncRNA NEAT1 sensitizes medulloblastoma cells to cisplatin through modulating the miR-23a-3p-glutaminase (GLS) axis. Bioengineered, 13(3), 7670-7682.

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Protein Extraction and Western Blot Analysis

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U20S cell pellets were lysed for 30 min on ice in 50 mM pH 8 Tris–HCl, 0.1% SDS, 350 mM NaCl, 0.25% Triton X-100 in the presence of Protease Inhibitor Cocktail 1X (Sigma). Then, samples were sonicated (at 30 s intervals) and centrifuged at 4°C for 10 min at 15 000 × g. The soluble portion of the extract was then quantified by Bradford assay (Bio-Rad) and loaded on SDS-Page gels. After gels running, proteins were transferred to a nitrocellulose membrane with a pore size of 0.45 μm (Ge Healthcare) using Trans-Blot^® Turbo TM device (Bio-Rad). Finally, the following primary antibodies were used: rabbit polyclonal anti-DDX3X A300-475A antibody (1:2000 dilution) (BETHYL), mouse 201055 anti-actin (1:1000 dilution) (Santa Cruz Biotechnology, Dallas, United States), mouse MAB3574 vinculin (1:10 000 dilution) (Millipore, Temecula, USA) and rabbit ab83943 anti-RNaseH2A (1:500 dilution) (Abcam, Cambridge, England).

Riva V., Garbelli A., Casiraghi F., Arena F., Trivisani C.I., Gagliardi A., Bini L., Schroeder M., Maffia A., Sabbioneda S, & Maga G. (2020). Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases. Nucleic Acids Research, 48(20), 11551-11565.

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