The biopsies were weighed and homogenized in a pre-cooled pestle and mortar with an appropriate amount of ice-cold PBS supplemented with 0.05% Tween 20 (Sigma-Aldrich, St Louis, Mo, USA) and 1% cocktail of protease inhibitors (Santa Cruz, California, USA). In brief, 1 μL of buffer added per 1 mg of the tissue, upon homogenization the content was centrifuged at 4000g for 20 min at 4 °C. Supernatants were aliquoted and stored at −80 °C. Total IgA was measured by Minineph™ human IgA kit (ZK010.R, Birmingham, UK) using Minieph nephelometry instrumentation (Binding Site, UK). We used sandwich ELISA kits for measurement of IgA subclasses, including IgA1 and IgA2 (MyBioSource, San Diego, USA) and for determination of the active form of TGF-β1 (R&D Systems, Minneapolis, USA) according to manufacturers’ instructions The minimum detectable amount of the applied kits was 28 ng/ml, 4 ng/ml, and 31 pg/ml for IgA1, IgA2, and TGF-β, respectively. The total protein of the extracts was measured with BCA method (Bio-Rad, Hercules, California) and the concentrations of total IgA, IgA1, IgA2, and TGF-β in the lysates were normalized to the protein concentrations.
Cocktail of protease inhibitors
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Cocktail of protease inhibitors is a reagent designed to inhibit a broad spectrum of protease enzymes commonly encountered in biological samples. This mixture of several protease inhibitors is intended to protect proteins from degradation during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
0.2 μm nitrocellulose membrane, by GE Healthcare (3 mentions)
Ecltm western blotting detect reagents, by GE Healthcare (3 mentions)
Bca method, by Abcam (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Tween 20, by Merck Group (1 mentions)
Bca method, by Bio-Rad (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Acrylamide gel, by Bio-Rad (1 mentions)
Phosphorylated akt ser473, by Cell Signaling Technology (1 mentions)
Pdgfrβ, by Cell Signaling Technology (1 mentions)
Ecl chemiluminescent kit, by GE Healthcare (1 mentions)
Antibodies against β actin, by Abcam (1 mentions)
Phosphorylated erk thr202 tyr204, by Cell Signaling Technology (1 mentions)
Anti pdgfrα, by Cell Signaling Technology (1 mentions)
Ripa assay buffer, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Fibronectin, by Merck Group (1 mentions)
E cadherin, by BD (1 mentions)
6 protocols using cocktail of protease inhibitors
1
Quantifying Mucosal Immunoglobulins and TGF-β
2
Western Blot Analysis of Signaling Proteins
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Cells and liver tissues were lysed or homogenized in RIPA buffer containing a cocktail of protease inhibitors (Santa Cruz, CA) according to the manufacturer's instruction. Protein extracts were loaded onto 12% acrylamide gels (Bio-Rad) and transferred onto nitrocellulose membranes. Protein bands were visualized with ECL-chemiluminescent kit (GE Healthcare, Piscataway, NJ). The antibodies against anti-PDGFRα, PDGFRβ, ERK, AKT, phosphorylated ERK (Thr202/Tyr204) and phosphorylated AKT (Ser473) were purchased from Cell Signaling Technology (Danvers, MA). GLI2 and GLI3 antibodies were purchased from Proteintech (Rosemont, IL). The antibodies against β-actin was purchased from Abcam (Cambridge, MA).
3
Protein Extraction and Western Blot Analysis from 3D Collagen Cultures
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Cells cultured in the 3D-collagen gel were recovered following enzymatic dissociation with collagenase 1 (2 mg/mL) and whole cell lysates were prepared by incubation with radioimmunoprecipitation (RIPA) assay buffer (Santa Cruz) supplemented with a cocktail of protease inhibitors (Santa Cruz) and the total protein concentrations were determined using bicinchoninic acid (BCA) method (BioVision). Then 30 µg of proteins were subjected to SDS-polyacrylamide gel electrophoresis before being electroblotted onto a 0.2 μm nitrocellulose membrane (GE Healthcare). After blocking with 5% nonfat dry milk in TBST [25 mmol/L Tris (pH, 7.4), 137 mmol/L NaCl, 0.5% Tween20], membranes were incubated at 4 °C overnight with following primary antibodies: p75 (1:1000, Cell Signaling), NOCTH3 (1:1000, Abcam), HES1(1:1000, Cell Signaling). GAPDH (1;2000, Cell Signaling) was used as a loading control. After extensively washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz), and blot signals were developed with ECLTM Western Blotting Detect Reagents (GE Health Care). All blots were derived from the same experiment and processed in parallel. Uncropped Western blotting images were provided in Supplementary Fig. 8 with the size markers labeled.
4
Western Blot Analysis of Stem Cell Markers
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Cell lysates were prepared by incubation with radioimmunoprecipitation assay buffer (Santa Cruz) supplemented with a cocktail of protease inhibitors (Santa Cruz) and the total protein concentrations were determined using bicinchoninic acid method (BioVision). Then 30 µg of proteins were subjected to SDS-polyacrylamide gel electrophoresis before being electroblotted onto a 0.2 μm nitrocellulose membrane (GE Healthcare). After blocking with 5% nonfat dry milk in TBST [25 mmol/l Tris (pH, 7.4), 137 mmol/l NaCl, 0.5% Tween20], membranes were incubated at 4 °C overnight with following primary antibodies: LGR5 (Invitrogen, PA5-35304), ALDH1 (BD Biosciences, 611194), OCT4 (Abcam, ab18976), β-catenin (Cell Signaling, 8480S), ABC (Cell Signaling, 8814S), cyclin A (Sigma, C4710), cyclin B (Sigma, C8831), cyclin D1 (Cell Signaling, 2926) and cyclin E (Cell Signaling, 4129), ZEB1 (Santa Cruz, sc-25388), fibronectin (Sigma, F3648), and E-Cadherin (BD Biosciences, 562869). β-actin (Santa Cruz, sc-47778) was used as loading control. After extensively washing, membranes were incubated with HRP-conjugated secondary antibodies (Santa Cruz) and blot signals were developed with ECLTM Western Blotting Detect Reagents (GE Health Care).
5
Western Blot Analysis of Cell Signaling
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HT-29 and SW480 cells were treated with or without DMC of IC50 for 72 h before cells were lysed in lysis buffer with 1 mM sodium fluoride, 2 mM sodium orthovanadate, and a cocktail of protease inhibitors (Santa Cruz, CA). For in vivo assay, fresh tumor tissues in RIPA lysis buffer containing 1 ug/ml PMSF were manually homogenized on ice using a glass homogenizer, then centrifuged at 10 000 g for 10 min to remove cellular and nuclear debris. Protein concentration was measured by modified Bradford assay. We electrophoresed 60 μg of total proteins on 10% SDS-PAGE gel and then transferred it to nitrocellulose membrane. The membranes were blocked with PBS containing 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with the primary antibodies against poly (ADP-ribose) polymerase (PARP), caspase-3, survivin, E-cadherin, and β-actin (Santa Cruz, CA). After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Beijing Zhongshan Co. Ltd.) at room temperature for 1 h, followed by chemiluminescence detection using a SuperSignal West Femto Maximum sensitivity substrate kit (Pierce). This process was independently repeated 3 times.
6
Astrocyte Protein Expression Analysis
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Planar-cultured GMSCs or astrocytes induced from GMSCs were harvested and whole-cell lysates were prepared by incubation with radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz) supplemented with a cocktail of protease inhibitors (Santa Cruz) and the total protein concentrations were determined using bicinchoninic acid (BCA) method (BioVision). Then 30 µg of proteins per well were subjected to SDS–polyacrylamide gel electrophoresis before being electroblotted onto a 0.2 μm nitrocellulose membrane (GE Healthcare). After blocking with 5% nonfat dry milk in TBST (25 mmol/L Tris, pH, 7.4, 137 mmol/L NaCl, 0.5% Tween20), membranes were incubated overnight at 4 °C with following primary antibodies: GFAP (1:1000, ab53554, Abcam), glutamine synthetase (1:1000, ab64613, Abcam), or GAPDH (1:2000, #5174, Cell Signaling) as loading control. After extensively washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz) and blot signals were developed with ECLTM Western Blotting Detect Reagents (GE Health Care) and scanned using Amersham Imager 680.
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