Protease inhibitor mixture tablet

Manufactured by Roche

Sourced in United States

Protease inhibitor mixture tablets is a laboratory product manufactured by Roche. The core function of this product is to inhibit the activity of proteases, enzymes that break down proteins. These tablets are commonly used in various biochemical and molecular biology applications that require the preservation of protein structures and activities.

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Lab products found in correlation

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Protease inhibitors (4287 citations) Protease inhibitor mixture (478 citations) Protease inhibitor tablet (349 citations)

26 protocols using protease inhibitor mixture tablet

Subcellular Protein Fractionation Protocol

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To fractionate proteins into cytoplasmic, nuclear, and chromatin-bound fractions, cells were grown in 6-cm dishes, scraped with PBS, and centrifuged (1000 rpm for 2 min). Input sample was lysed in SNTBS. For fractionation purposes, a separate sample of cells was lysed with buffer containing 10 mm HEPES, 10 mm KCl, 1.5 mm MgCl₂, 10% glycerol, 340 mm saccharose, 1 mm DTT, protease inhibitor mixture tablet (Roche Applied Science), 0.1% Triton for 8 min on ice. After centrifugation (13,700 rpm for 5 min), the supernatant was taken as the cytosolic fraction. To obtain the nuclear fraction, the pellet from the previous centrifugation step was lysed in buffer containing 3 mm EDTA, 200 μm EGTA, 1 mm DTT, protease inhibitor mixture tablet (Roche Applied Science). After centrifugation (4000 rpm for 4 min), the supernatant was taken as the nuclear fraction, and the pellet was lysed in SNTBS buffer containing additional 3 mm EDTA, 200 μm EGTA, 1 mm DTT, protease inhibitor mixture tablet (Roche Applied Science), and taken as the chromatin-bound fraction. Equal amounts of protein from each fraction, including the input lysate, were subsequently separated by SDS-PAGE and analyzed by Western blotting.

Sha Z., Blyszcz T., González-Prieto R., Vertegaal A.C, & Goldberg A.L. (2019). Inhibiting ubiquitination causes an accumulation of SUMOylated newly synthesized nuclear proteins at PML bodies. The Journal of Biological Chemistry, 294(42), 15218-15234.

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ICAM-1 Clustering Assay in HUVECs

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TNFα-stimulated HUVEC monolayers (10-cm-dish), transfected with si-Control or si-CD2AP, were incubated with 45 μl beads coated (4×10⁵ beads/μl) with anti-ICAM-1 antibody (see above) for indicated times at 37°C and 5% CO₂ to induce ICAM-1 clustering. Cells were washed with PBS containing 1 mM CaCl₂ and 0.5 mM MgCl₂ and lysed in 50 mM Tris pH 7.4, 500 mM NaCl, 0.5 mM MgCl₂, 1% Triton-X100, 0.5% deoxycholic acid, 0.1% SDS supplemented with protease inhibitor mixture tablets (Roche). To measure Rac1-GTP levels, lysates were centrifuged (10,000g, 10 min, 4°C) and supernatant was incubated with Streptavidin agarose beads (Sigma) and 30 μg biotinylated peptide encoding the CRIB domain of the effector PAK1 (synthesized using Fmoc-solid-phase chemistry, Netherlands Cancer Institute Amsterdam) for 30 min at 4°C under continuous mixing (19 (link)). Beads were washed four times in 50 mM Tris, 150 mM NaCl, 0.5 mM MgCl₂, 1% Triton-X100, pH 7.4 supplemented with protease inhibitor mixture tablets (Roche) and resuspended in SDS-sample buffer and analyzed using western blotting.

Schaefer A., van Duijn T.J., Majolee J., Burridge K, & Hordijk P.L. (2017). Endothelial CD2AP binds the receptor ICAM-1 to control mechanosignaling, leukocyte adhesion and the route of leukocyte diapedesis in vitro. Journal of immunology (Baltimore, Md. : 1950), 198(12), 4823-4836.

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Affinity Purification of Bcl-xL and HBx Interactions

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HepG2 cells were lysed in lysis buffer [100 mM NaCl, 0.5 mM MgCl₂, 0.15 mM CaCl₂, 1% (v/v) Nonidet P-40, 10 mM Tris-HCl, pH 8.0] containing protease inhibitor mixture tablets (Roche). Cell lysate was centrifuged at 12,000 × g for 10 min at 4 °C to removed debris and precleared with magnetic beads conjugated with streptavidin (Dynabeads® M-280 Streptavidin, Invitrogen). Subsequently, the precleared supernatant was incubated with C-terminal biotinylated peptides for 1 h with gentle shaking at 4 °C. A new batch of streptavidin beads were added and incubated for another 2 h. The beads were washed five times with the lysis buffer and the bound proteins were resolved on a 13.5% sodium dodecyl sulfate-polyacrylamide gel and detected by anti-Bcl-xL antibody (Cell Signaling Technology).
Similarly, HepG2 cells transfected with pTT22m-HBx-WT or pTT22m-HBx-(W120A/L123A) plasmid were lysed. Co-IP experiments were performed using an anti-HBx antibody (16F9) and Dynabeads Protein G for Immunoprecipitation (Thermo Fisher Scientific, catalog no. 10004D).

Zhang T.Y., Chen H.Y., Cao J.L., Xiong H.L., Mo X.B., Li T.L., Kang X.Z., Zhao J.H., Yin B., Zhao X., Huang C.H., Yuan Q., Xue D., Xia N.S, & Yuan Y.A. (2019). Structural and functional analyses of hepatitis B virus X protein BH3-like domain and Bcl-xL interaction. Nature Communications, 10, 3192.

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Protein Interaction and Modification Analysis

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Anti-EGFR, GAPDH and 14-3-3ζ were from Santa Cruz Biotechnology. Anti-Pan-AKT, anti-AKT pSer⁴⁷³, anti phosphoEGFR antibodies were from Cell Signaling Technology. HA-peroxidase and anti-PTP1B (FG6) was from Millipore. PT-66-agarose-conjugated beads, anti-FLAG M2 beads, and anti-HA beads and anti-Flag M2 peroxidase were purchased from Sigma. Anti-PTP1B pSer⁵⁰ (Ab62320) were from Abcam. Streptavidin-HRP was from GE Healthcare. HRP-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. Protease inhibitor mixture tablets were from Roche. Catalase and superoxide dismutase were from Calbiochem. Surfact-Amps Nonidet P-40, zeba desalt spin columns, EZ-Link biotin-iodoacetyl-PEG2 (biotin-IAP), and iodoacetic acid were from ThermoScientific. The pTyr loop-derived peptide (CKNRNRYRDVS) and phospho-Ser⁵⁰ pTyr loop-derived peptide (CKNRNRYRDVpS) were from GenScript USA Inc. BIACore sensor NTA and Streptavidin chips were from GE healthcare.

Londhe A.D., Bergeron A., Curley S.M., Zhang F., Rivera K.D., Kannan A., Coulis G., Rizvi S.H., Kim S.J., Pappin D.J., Tonks N.K., Linhardt R.J, & Boivin B. (2019). Regulation of PTP1B activation through disruption of redox-complex formation. Nature chemical biology, 16(2), 122-125.

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SHP2 Phosphatase Activity Assay

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SHP2 activity was analyzed using a Human Active SHP-2 kit (R&D Systems Inc., Minneapolis, MN, USA). Briefly, cells were lysed in a lysis buffer ([50 mM HEPES, 0.1 mM EGTA, 0.1 mM EDTA, 120 mM NaCl, 0.5-% Nonidet-P40 [NP-40], pH 7.5 supplemented with fresh protease-inhibitor-mixture tablets (Roche Applied Science). The SHP2 proteins were then immunoprecipitated using active SHP2 immunoprecipitation beads (R&D Systems Inc.), and washed 3 times in the lysis buffer and 4 times in a phosphatase assay buffer (10 mM HEPES, 0.1 mM EGTA, 0.1 mM EDTA, 0.5-% BSA, 1 mM dithiothreitol [DTT], pH 7.5). The phosphatase reaction was initiated by incubating the immunocomplexes for 30 min at 37°C in the presence of tyrosine phosphatase substrate I, DADEY (PO3) LIPQQG, according to the manufacturer's instructions. Phosphatase activity was determined using a microplate reader (SpectraMax 190 Absorbance Microplate Reader; Molecular Devices) at 620 nm.

Wang H.C., Chiang W.F., Huang H.H., Shen Y.Y, & Chiang H.C. (2014). Src-homology 2 domain-containing tyrosine phosphatase 2 promotes oral cancer invasion and metastasis. BMC Cancer, 14, 442.

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Quantifying Bacterial Load and Host Response

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To determine bacterial burden, lungs, livers and spleen were harvested in PBS, homogenized with glass homogenizer, and plated on TSA II plates in four serial dilutions. Colonies were counted after overnight incubation at 37°C, 5%CO2. To determine gene expression, lungs and livers were placed in TRIzol reagent (Invitrogen), homogenized and processed according to manufacturer’s protocol. One microgram of RNA was used to synthesize cDNA. Real-time PCR primers with SSOFast Probes Supermix probes were used. Threshold cycle (C_T) values were normalized to Hprt. For protein analysis, tissues were placed in RIPA cell lysis buffer with protease inhibitor mixture tablets (Roche Applied Science) homogenized, and centrifuged at 10,000 3 g. IL-22 levels in supernatants were measured by ELISA (eBioscience) and normalized to total protein levels (Pierce BCA Protein Assay Kit; Thermo Fisher Scientific).

Trevejo-Nunez G., Elsegeiny W., Conboy P., Chen K, & Kolls J.K. (2016). Critical role of IL22/IL22RA1 signaling in pneumococcal pneumonia. Journal of immunology (Baltimore, Md. : 1950), 197(5), 1877-1883.

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Western Blot Protein Expression Analysis

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For Western blots, cells were transfected on 6-well polystyrene plates (Corning). 24 h after transfection, the cells were washed twice with ice-cold PBS followed by scraping and extraction on ice for 30 min in a buffer containing 10 mM Tris-HCl, pH 6.8, 1 mM EDTA, 150 mM NaCl, 0.25% Nonidet P-40, 1% Triton X-100, 1 µM NaF and protease inhibitor mixture tablets (Roche Applied Science). Cell debris was removed by centrifugation at 16,000× g. The protein concentrations were determined using the BCA protein assay kit (Pierce/Thermo) and equal amounts of total protein (25–40 µg) per lane were resolved in a 4–12% gradient Bis-Tris gels (Novex, Invitrogen) under reducing conditions. After transfer to PVDF membranes (Amersham Biosciences/GE Healthcare), the filters were probed with the following antibodies: A8717 (Sigma; APP C-terminus), dNGluc (Proteintech Group Inc) and GAPDH (Millipore). After incubation with horseradish-conjugated secondary antibodies, the signal was developed using ECL Western blotting detection reagent (Pierce/Thermo).

Merezhko M., Muggalla P., Nykänen N.P., Yan X., Sakha P, & Huttunen H.J. (2014). Multiplex Assay for Live-Cell Monitoring of Cellular Fates of Amyloid-β Precursor Protein (APP). PLoS ONE, 9(6), e98619.

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Protein Interaction and Modification Analysis

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Western Blotting of E-Cadherin in EOC Cells

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For western blotting of E-cadherin, EOC cells were plated in 10-cm dishes. On the following day, the cells were grown to 70–80% subconfluency and treated with lysis buffer containing 1% Triton X-100 in PBS and protease inhibitor mixture tablets (Roche, Barcelona, Spain). For the TGF-β1-treated cells, total cell lysates were isolated in the same way after a 96-h incubation with TGF-β1.
Samples were electrophoresed on a 10% SDS-polyacrylamide gel and transferred electrophoretically to Immobilon membranes (Millipore, Bedford, MA, USA). After blocking in blocking solution (5% nonfat dry milk/0.1% Tween-20/PBS), the membranes were incubated overnight with a recommended dilution of primary antibodies. We used the following antibodies: anti-E-cadherin (Santa Cruz Biotechnology, Dallas, TX, USA) and anti-β-actin (Sigma-Aldrich). The primary antibodies were washed in 0.05% Tween-20/PBS and then incubated with horseradish peroxidase-conjugated secondary antibody. Immunoreactive proteins were stained using a chemiluminescence detection system (ECL; Amersham, Arlington Heights, Il, USA). An Ab against β-actin (AC-15; Sigma-Aldrich) was used to standardize the protein loading.

UTSUMI F., KAJIYAMA H., NAKAMURA K., TANAKA H., MIZUNO M., TOYOKUNI S., HORI M, & KIKKAWA F. (2016). Variable susceptibility of ovarian cancer cells to non-thermal plasma-activated medium. Oncology Reports, 35(6), 3169-3177.

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Peptide-based Binding Assay for ICAM-1 and VCAM-1

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N-terminal biotinylated peptides encode the intracellular domain of human ICAM-1 or VCAM-1 were synthesized using Fmoc-solid-phase chemistry (Netherlands Cancer Institute Amsterdam). To analyze binding to these peptides, a TNFα-stimulated HUVEC monolayer in a 10-cm-dish was washed with PBS containing 1 mM CaCl₂ and 0.5 mM MgCl₂ and lysed for 10 min on ice in NP40-buffer (25 mM Tris, 100 mM NaCl, 10 mM MgCl₂, 10% glycerol, 1% NP40, pH 7.4), supplemented with a phosphatase inhibitor cocktail (Sigma) and protease inhibitor mixture tablets (Roche). After cell lysis and centrifugation (10,000 g, 10 min, 4°C), supernatant was incubated with Streptavidin agarose beads (Sigma) and 5 μg biotinylated peptide for 3 h at 4°C under continuous mixing (16 (link)). Empty beads were used as control. Beads were washed five times with NP40-buffer, resuspended in SDS-sample-buffer. Proteins were detected using western blotting. To analyze the binding of the CD2AP constructs to the cytoplasmic ICAM-1 domain, Hela cells were transfected with CD2AP-GFP, N-CD2AP-GFP, C-CD2AP-GFP or GFP and pull-down studies with the N-terminal biotinylated peptide encode the intracellular domain of human ICAM-1 were performed as described above.

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