Complete mini protease inhibitor tablet

Manufactured by Roche

Sourced in United States, United Kingdom, Switzerland, Germany, Denmark

The Complete mini protease inhibitor tablet is a laboratory reagent designed to inhibit the activity of proteases, which are enzymes that break down proteins. It is a concentrated mixture of protease inhibitors formulated into a compact tablet form for convenient use in various biochemical and cell-based assays.

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Lab products found in correlation

β actin, by Merck Group (12 mentions) Cell lysis buffer, by Thermo Fisher Scientific (7 mentions) Horseradish peroxidase conjugated anti rabbit or anti mouse igg, by Jackson ImmunoResearch (7 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions) Cell lysis buffer, by Cell Signaling Technology (4 mentions) Dc protein assay, by Bio-Rad (4 mentions) Immobilon pvdf membrane, by Merck Group (3 mentions) Elisa, by R&D Systems (3 mentions) Phosstop, by Roche (3 mentions) Phosphatase inhibitor cocktail 2, by Merck Group (3 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (3 mentions) Dynabead, by Thermo Fisher Scientific (3 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Bio plex manager software, by Bio-Rad (2 mentions) Complete mini tablet, by Roche (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Polyacrylamide gel, by Bio-Rad (2 mentions) Radioimmunoprecipitation assay buffer, by Merck Group (2 mentions) Bradford assay, by Bio-Rad (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)

91 protocols using complete mini protease inhibitor tablet

Protein Extraction from Frozen Tissue

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Proteins were extracted as previously described [17] (link), but with modifications. In short frozen pulverized tissue was homogenized in ice-cold Tris-EDTA (50 mM Tris, 1 mM EDTA in water added a complete mini protease inhibitor tablet (Roche Diagnostics, Hvidovre, Denmark) pH 7.4) with a polytron. Following centrifugation at 15.300×g for 20 min at 4°C, the supernatant was discarded and the pellet was dissolved in SDS sample buffer (125 mM Tris, 4% SDS, 20% glycerol in water added complete mini protease inhibitor tablet (Roche Diagnostics, Hvidovre, Denmark) pH 6.8). Following centrifugation at 15.300×g for 90 min at 4°C, the supernatant was collected, and protein concentration was measured using the Bradford method with BSA as standards.

Rahbek M., Nazemi S., Ødum L., Gupta S., Poulsen S.S., Hay-Schmidt A, & Klaerke D.A. (2014). Expression of the Small Conductance Ca2+-Activated Potassium Channel Subtype 3 (SK3) in Rat Uterus after Stimulation with 17β-Estradiol. PLoS ONE, 9(2), e87652.

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Immunoprecipitation of GFP and ATXN3 in HEK293 Cells

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Human embryonic kidney 293 (HEK293) cells were cultured in DMEM, supplemented with 10% FBS, 100U/ml penicillin/streptomycin. Transfections were carried out with lipofectamine 2000 (invitrogen) as described previously (Zeng et al., 2012 (link)). Transfected cells were lysed with 1% Triton X-100 lysis buffer containing 150 mM NaCl, 20 mM Tris/HCl, pH 8.0, 5 mM EDTA, 2 mM N-ethylmaleimide (Life technologies) and complete Mini Protease Inhibitor tablets (Roche). Nuclei and insoluble material were removed by centrifugation. Antigen-antibody complexes were immunoprecipitated with anti-GFP (Invitrogen) or anti-ATXN3 1H9 and protein G beads (Sigma) at 4°C overnight with rotation. After washing three times in lysis buffer, the immunoprecipitates were dissolved in SDS loading buffer and loaded on 10% SDS/PAGE and transferred to polyvinylidene difluoride membranes. The membranes were incubated with the following antibodies: rabbit anti-GFP (1:1000; Santa Cruz biotechnology), rabbit anti-tubulin-α (1:10,000; cell signaling), rabbit anti-flag (1: 5000; Sigma), mouse anti-ATXN3 1H9 (1:1000; Millipore). After incubation with secondary antibodies, blots were developed with blue basic autorad films (GeneMate).

Zeng L., Wang B., Merillat S.A., Minakawa E., Perkins M.D., Ramani B., Tallaksen-Greene S.J., do Carmo Costa M., Albin R.L, & Paulson H.L. (2015). Differential recruitment of UBQLN2 to nuclear inclusions in the polyglutamine diseases HD and SCA3. Neurobiology of disease, 82, 281-288.

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Western Blot Analysis of p53 and p21

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Cells were lysed in RIPA lysis buffer (50 mM Tris/HCl, pH 8.0, 250 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, complete mini protease inhibitor tablets (Roche)). Lysates were sonicated and centrifuged at 16,060 g for 15 min at 4°C. 20 μg of whole cell lysate per lane were separated using 10% SDS-acrylamide gels and transferred on Immobilon PVDF membranes (Millipore). Antibodies used were specific for p53 (DO-1), p21 (BD Pharmingen) and β-actin (Sigma, A2066).

Kaller M., Götz U, & Hermeking H. (2017). Loss of p53-inducible long non-coding RNA LINC01021 increases chemosensitivity. Oncotarget, 8(61), 102783-102800.

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Cell Lysis and Western Blot Analysis

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Lysates were prepared from cells treated for 24 h. Harvested cells were lysed in 20 mmol/L Tris pH 7.7, 100 mmol/L NaCl, 20 mmol/L β-glycerophosphate, 5 mmol/L MgCl2, 1 mmol/L NaF, 0.1% Triton X-100, 5% glycerol plus freshly added 1 mmol/L DTT, 0.1 µmol/L okadaic acid, 1 mmol/L sodium vanadate, and cOmplete Mini Protease Inhibitor Tablets (Roche, #11836170001). For SDS-PAGE, equal amounts of protein/lane were loaded. After gel transfer onto PDV membrane, immunoblots were performed using the following antibodies: CDK9 (Cell Signalling Technology #C12FT; 1:1,000), RNA polymerase II (Abcam #ab5131; 1:1000), MCL-1 (Abcam #Y37; 1:10omogeniospho-Rb (Cell Signalling Technology #d2b12; 1:1,000), c-Myc (Abcam #YB69; 1:1000). Secondary antibodies were from Cell Signalling Technology (#7076 or #7074). ECL detection was performed.

Yang B., Quan Y., Zhao W., Ji Y., Yang X., Li J., Li Y., Liu X., Wang Y, & Li Y. (2023). Design, synthesis and biological evaluation of 2-((4-sulfamoylphenyl)amino)-pyrrolo[2,3-d]pyrimidine derivatives as CDK inhibitors. Journal of Enzyme Inhibition and Medicinal Chemistry, 38(1), 2169282.

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Spinal Cord Fractionation Protocol

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Mice were sacrificed at the age of 97 or 110 days. Their spinal cords were collected and homogenized on ice in 5 volumes (w/v) of T-per extraction buffer (Pierce, USA) complemented with protease inhibitor tablets (Complete Mini Protease Inhibitor Tablets, Roche) and phosphatase inhibitor cocktail tablets (phosSTOP, Roche). After sonication homogenates were centrifuged at 100,000g for 1hr at 4°C. The resulting supernatants represent the soluble fraction.
The pellets were resuspended in T-per extraction buffer, containing protease and phosphatase inhibitors, 0.5% TritonX- 100, 1% sodium deoxycholate and 3% SDS, sonicated and centrifuged at 20,000g for 10 min at 4°C. The resulting supernatants represent the membrane fraction.

Rabinovich-Toidman P., Rabinovich-Nikitin I., Ezra A., Barbiro B., Fogel H., Slutsky I, & Solomon B. (2015). Mutant SOD1 Increases APP Expression and Phosphorylation in Cellular and Animal Models of ALS. PLoS ONE, 10(11), e0143420.

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Whole-Cell Protein Extraction and Western Blotting

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Briefly, obtain whole cell lysate by applying 1% NP40 buffer (1% NP40 detergent with 150 mM NaCl, 50 mM Tris [pH 8], 1 mM dithiothreitol, plus 1× Roche cOmplete Mini protease inhibitor tablets [cat. # 11836170001] and 1% each of Sigma phosphatase inhibitor cocktail #s 2 [cat. # P5726] and 3 [cat. # P0044]) to a pellet of collected cells. Use 150 µl of lysis buffer for mostly confluent cells in a 35 mm well, or 300 µl for cells in a 10 cm dish (providing a more concentrated lysate). Separate the proteins by running the lysate on a polyacrylamide gel (Bio-Rad, cat. # 345-0043), transfer to nitrocellulose membrane (Bio-Rad, cat. # 162-0112), and blot for the protein of interest using standard molecular biology techniques.

Satterstrom F.K, & Haigis M.C. (2014). Luciferase-based reporter to monitor the transcriptional activity of the SIRT3 promoter. Methods in enzymology, 543, 141-163.

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Western Blotting of Transfected Cell Lysates

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K562 cells were transfected with lipofectamine 2000 according to the manufacturer’s instructions. Normal mouse bone marrow cells were isolated fresh on the day of the experiment. CSF3R^T618I/CEBPA^V314VW cells were cultured in IMDM media supplemented with 20% FCS after isolation from methocult. In all cases, 2 × 10⁶ cells were used per condition. Cells were lysed in cell lysis buffer (Cell Signaling Technologies) containing complete mini protease inhibitor tablets (Roche). To pellet cellular debris, the lysates were spun at 12,000 r.p.m., 4 °C for 10 min, and subsequently mixed with 3× SDS sample buffer (75 mmol/L Tris (pH 6.8), 3% SDS, 15% glycerol, 8% β-mercaptoethanol, and 0.1% bromophenol blue). Samples were incubated at 95 °C for 5 min and run on Criterion 4 to 15% Tris-HCl gradient gels (Bio-Rad). Gels were transferred to PVDF membranes and blocked in Tris-buffered saline with Tween (TBST) with 5% bovine serum albumin (BSA). Blots were probed with the antibodies listed in Supplementary Table 3. Horseradish peroxidase-conjugated secondary antibodies against mouse IgG and rabbit IgG (CS) were used followed by imaging of the blots on a BioRad Touch Gel Doc (BioRad).

Braun T.P., Okhovat M., Coblentz C., Carratt S.A., Foley A., Schonrock Z., Curtiss B.M., Nevonen K., Davis B., Garcia B., LaTocha D., Weeder B.R., Grzadkowski M.R., Estabrook J.C., Manning H.G., Watanabe-Smith K., Jeng S., Smith J.L., Leonti A.R., Ries R.E., McWeeney S., Di Genua C., Drissen R., Nerlov C., Meshinchi S., Carbone L., Druker B.J, & Maxson J.E. (2019). Myeloid lineage enhancers drive oncogene synergy in CEBPA/CSF3R mutant acute myeloid leukemia. Nature Communications, 10, 5455.

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Quantifying STAT1 and C/EBPβ in Mtb-Infected CD34+ Cells

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CD34⁺ cells were seeded at 3 × 10⁵ cells in 24-well plate and infected with Mtb (MOI3). After 5 days of infection, cells were centrifuged at 4°C, pellet was lysed using M-PER lysis buffer (Thermo Fisher Scientific) containing protease inhibitors (Complete, Mini Protease Inhibitor Tablets, Roche) and protein extracts were prepared according to manufacturer’s instructions. For Western blot, 15 µg of total protein were separated and transferred to nitrocellulose difluoride 0.22 µm blotting membranes. Membranes were blocked for 1 hr with TBST containing 5% w/v BSA and subsequently washed three times with TBST for 5 min each wash. Further, membranes were then probed with anti-pSTAT1 Y701 1:1000 (M135 – Abcam), anti-STAT1 1:1000 (SM1 – Abcam), anti-C/EBPβ 1:250 (sc-150 – Santa Cruz) or anti-β-actin 1:5000 (8226 – Abcam) primary antibodies diluted in 5% w/v BSA, 0.1% tween 20 in TBS, at 4°C with gentle shaking overnight. Membranes were washed with TBST, incubated in secondary HRP-linked Ab for 2 hr at room temperature, washed and chemiluminescence developed using ECL substrate (Pierce). Relative expression was normalized with β-actin control and pixel area was calculated using ImageJ software.

Delgobo M., Mendes D.A., Kozlova E., Rocha E.L., Rodrigues-Luiz G.F., Mascarin L., Dias G., Patrício D.O., Dierckx T., Bicca M.A., Bretton G., Tenório de Menezes Y.K., Starick M.R., Rovaris D., Del Moral J., Mansur D.S., Van Weyenbergh J, & Báfica A. (2019). An evolutionary recent IFN/IL-6/CEBP axis is linked to monocyte expansion and tuberculosis severity in humans. eLife, 8, e47013.

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Western Blot Analysis of UCP1 in BAT

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BAT samples were homogenized in ice‐cold radioimmunoprecipitation assay buffer (Sigma‐Aldrich, St. Louis, Missouri) supplemented with complete Mini Protease Inhibitor Tablets (Roche Diagnostics, Mannheim, Germany) and PhosSTOP Phosphatase Inhibitor Cocktail Tablets (Roche Diagnostics). Total protein was collected from the fat pads after being centrifuged at 12,000 rpm at 4°C for 30 minutes. After determining total protein by the Bradford protein assay, equal amounts (30 μg) of protein for each group were resolved on 4% to 20% TGX stain‐free gels (Bio‐Rad Laboratories GmbH, Munich, Germany) and transferred onto polyvinylidene difluoride membranes. The membranes were probed with the primary antibody UCP1 (U6382; Sigma‐Aldrich) in 0.01M Tris‐buffered saline supplemented with Triton X‐100 (Sigma‐Aldrich Sweden AB, Stockholm, Sweden) containing 5% nonfat dry milk overnight followed by horseradish peroxidase‐conjugated secondary antibody. Ultraviolet activation of the stain‐free gel on a ChemiDoc MP Imaging System (Bio‐Rad Laboratories) was used to control for proper loading as previously described 21, 22. Band densitometry and quantification were performed using Image Laboratory (version 5.0; Bio‐Rad Laboratories AB, Solna, Sweden). The protein band densities were normalized to the total‐protein loading control. The analysis was performed as described previously 23.

Rabasa C., Askevik K., Schéle E., Hu M., Vogel H, & Dickson S.L. (2019). Divergent Metabolic Effects of Acute Versus Chronic Repeated Forced Swim Stress in the Rat. Obesity (Silver Spring, Md.), 27(3), 427-433.

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Quantifying Lung Cytokine and Chemokine Profiles

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The right lung was homogenized in PBS supplemented with Complete Mini protease inhibitor tablets (Roche), clarified by centrifugation and stored at −80 °C. Supernatants from lung homogenates were analyzed for protein levels of 32 cytokines and chemokines using a Milliplex multiplex suspension cytokine array (Millipore) according to the manufacturer’s instructions. The data were analyzed using Bio-Plex Manager software (Bio-Rad Laboratories). IL-33, CCL17 and CCL22 levels were quantified by ELISA (R&D Systems). In the studies examining the effects of IL-7R blockade, proallergic chemokine levels were analyzed in bronchoalveolar lavage.

Reeder K.M., Dunaway C.W., Blackburn J.P., Yu Z., Matalon S., Hastie A.T., Ampleford E.J., Meyers D.A, & Steele C. (2018). The common γ-chain cytokine IL-7 promotes immunopathogenesis during fungal asthma. Mucosal immunology, 11(5), 1352-1362.

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