For Western blot analysis, cells were seeded in a 6-well plate at a seeding density of 1 × 106 cells/well and treated with curcumin concentrations for 48 h. Cells treated with 0.1% DMSO were used as a negative control. Cells were lysed in ice-cold RIPA buffer supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich, Gillingham, UK). All samples were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) nitrocellulose membrane (Merck Millipore, Darmstadt, Germany). The membrane was blocked with 5% BSA in Tris-buffered saline supplemented with 0.1% Tween-20 buffer (Sigma-Aldrich, Gillingham, UK). Western blot analyses were performed using primary antibodies against p-NF-κB (#3033), p-P70S6K (#9204), cleaved caspase-3 (#9661), p-Akt (#4060) and β-actin (#3700) (Cell Signaling Technology, Danvers, MA, USA). The signals were detected using AmershamTM ECLTM Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, Amersham, UK). Protein bands were visualized by Molecular Imager ChemiDocTM XRS+ Imaging System (Bio-Rad, Hercules, CA, USA) and analyzed by Image Lab software (version 6.0) (Bio-Rad, Hercules, CA, USA).
Tween 20 buffer
Manufactured by Merck Group
Sourced in United States
Tween-20 is a non-ionic detergent commonly used as a buffer in various laboratory applications. It is a polyoxyethylene (20) sorbitan monolaurate. Tween-20 is a versatile, water-soluble, and non-denaturing agent used to facilitate the solubilization and stabilization of proteins and other biomolecules in aqueous solutions.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Sds polyacrylamide gel, by Bio-Rad (2 mentions)
Immobilon western, by Merck Group (2 mentions)
Tris buffered saline (tbs), by Merck Group (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Image lab software version 6, by Bio-Rad (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Molecular imager chemidoc xrs imaging system, by Bio-Rad (1 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
Amersham ecltm prime western blotting detection reagent, by GE Healthcare (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
P nf κb, by Cell Signaling Technology (1 mentions)
Alexa fluor 647 protein labeling kit, by Thermo Fisher Scientific (1 mentions)
Acetate buffer, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Cytiva (1 mentions)
Gel pro analyzer 3, by Media Cybernetics (1 mentions)
Gel pro analyzer software, by Media Cybernetics (1 mentions)
4 protocols using tween 20 buffer
1
Curcumin Modulates Signaling Pathways in Cells
2
Multistep Antibody Functionalization for Protein Detection
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PSA, monoclonal PSA capture antibody (c-Ab), and polyclonal PSA detection antibody (d-Ab) were ordered from SCIPAC Ltd. (Sittingbourne, UK). The triethylene glycol mono-11-mercaptoundecylether (thiol-PEG, #673110), phosphate-buffered saline (PBS) buffer tablets, and Tween-20 buffer were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The thiol-PEG7 acid (Thiol-COOH, #37156-0795) was purchased from Polypure AS (Oslo, Norway). 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were purchased from TCI (Tokyo, Japan). Alexa Fluor 647 protein labeling kit (A-20173) was purchased from Invitrogen (Singapore) for labeling the d-Ab at a dye-to-protein ratio of ~4. Acetate buffer (10 mM, pH 5.0) was prepared by suitable mixing of sodium acetate and acetic acid that were obtained from Sigma-Aldrich Co.
3
Quantification of Oxidative Phosphorylation Complexes
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A 30-μg aliquot of whole-cell lysate was fractionated on a 12% SDS–polyacrylamide gel (Bio-Rad Laboratories, Richmond, CA, USA) and blotted onto a polyvinylidene difluoride membrane (Amersham Biosciences, Buckinghamshire, UK). Nonspecific binding was blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 buffer (Sigma-Aldrich), and the membrane was incubated with a cocktail of monoclonal antibodies (1:800 dilution) purchased from Mitosciences (Eugene, OR, USA) for evaluation of oxidative phosphorylation complexes, including complex I (CI)-20 kDa subunit (NDUFB8), complex II (CII)-30 kDa subunit (SDHB), complex III-core 2 subunit (CIII)-48 kDa, complex IV-subunit I (CIV-I)-40 kDa, and complex V-subunit alpha (CV)-55 kDa. After incubation with a horseradish peroxidase-conjugated secondary antibody (1:50,000 dilution; Jackson ImmunoResearch), protein intensity was determined using an enhanced chemiluminescence reagent (Immobilon Western, Millipore). Bands were digitally scanned, and intensities were quantified using Gel-PRO Analyzer 3.0 software (Media Cybernetics, Rockville, MD, USA). Immunoreactivity of rabbit anti-glyceraldehyde-3-phosphate dehydrogenase antibodies (GADPH) (1:1000 dilution, Santa Cruz Biotechnology) was analyzed simultaneously as an internal control.
4
Quantification of Oxidative Phosphorylation Complexes
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An 30-μg aliquot of whole-cell lysate was fractionated on a 12% SDS-polyacrylamide gel (Bio-Rad Laboratories, Richmond, CA, USA) and blotted onto a polyvinylidene di uoride membrane (Amersham Biosciences, Buckinghamshire, UK). Nonspeci c binding was blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 buffer (Sigma-Aldrich), and the membrane was incubated with a cocktail of monoclonal antibodies (1:800 dilution) purchased from Mitosciences (Eugene, OR, USA) for evaluation of oxidative phosphorylation complexes, including complex I (CI)-20 kDa subunit (NDUFB8), complex II (CII)-30 kDa subunit (SDHB), complex III-core 2 subunit (CIII)-48 kDa, complex IV-subunit I (CIV-I)-40 kDa, and complex V-subunit alpha (CV)-55 kDa. After incubation with a horseradish peroxidase-conjugated secondary antibody (1:50,000 dilution; Jackson ImmunoResearch), protein intensity was determined using an enhanced chemiluminescence reagent (Immobilon Western, Millipore). Bands were digitally scanned, and intensities were quanti ed using Gel-PRO Analyzer software (Media Cybernetics, Rockville, MD, USA). Immunoreactivity of rabbit anti-glyceraldehyde-3-phosphate dehydrogenase antibodies (GADPH) (1:1000 dilution, Santa Cruz Biotechnology) was analyzed simultaneously as an internal control.
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