Proteins were extracted with RIPA buffer containing an inhibitor cocktail (Roche, Sussex, UK) and protein concentration was determined using a Bradford dye-based assay (Bio-Rad, Hemel Hempstead, UK). Total protein (30 μg) was subjected to SDS–PAGE followed by immunoblotting with appropriate antibodies at the recommended dilutions. The blots were then incubated with peroxidase-linked secondary antibodies followed by enhanced-chemiluminescent detection using the Super Signal Chemiluminescence Kit (Thermo scientific). The following antibodies were used: PGC-1α (rabbit, 1 : 1000, Abcam, Cambridge, UK), MEF2 (rabbit, 1 : 500, Santa Cruz, Middlesex, UK), Phospho-S6 Ribosomal Protein (Ser235/236) (rabbit, 1 : 100, Cell Signaling Technology, UK), S6 Ribosomal Protein (5G10) (Rabbit, 1 : 100, Cell Signaling Technology), TFAM (rabbit, 1 : 100, Sigma, UK), NRF1 (rabbit, 1 : 500, Life Span Bioscience, Nottingham, UK), Cleaved caspase-3 (rabbit, 1 :1 000, Cell Signaling), GAD67 (mouse, 1 : 1000, Millipore, Watford, UK), MitoProfile Total OXPHOS Rodent WB Antibody Cocktail (mouse, 1 : 250, Abcam, UK), LC3B (rabbit, 1 : 1000, Sigma), β-Tubulin (rabbit, 1 : 1000, Santa Cruz), Synapsin (1 : 1000, Synaptic System, Goettingen, Germany), Actin (1 : 5000, Sigma), Cyclophillin D (1 : 1000, Abcam), GLS2 (1 : 100, Abcam), PFKp (1 : 500, Abcam) and GADPH (mouse, 1 : 10 000, Sigma).
Bradford dye based assay
Manufactured by Bio-Rad
Sourced in United Kingdom, Italy
The Bradford dye-based assay is a colorimetric method for the quantitative determination of protein concentration. It is based on the binding of the dye Coomassie Brilliant Blue G-250 to proteins, which results in a color change that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal chemiluminescence kit, by Thermo Fisher Scientific (2 mentions)
Gad67, by Merck Group (1 mentions)
Mitoprofile total oxphos rodent wb antibody cocktail, by Abcam (1 mentions)
Pgc 1α, by Abcam (1 mentions)
Gadph, by Merck Group (1 mentions)
Phospho s6 ribosomal protein ser235 236, by Cell Signaling Technology (1 mentions)
S6 ribosomal protein 5g10, by Cell Signaling Technology (1 mentions)
β tubulin, by Santa Cruz Biotechnology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Qiashredder, by Qiagen (1 mentions)
Anti actin, by Merck Group (1 mentions)
Ecl kit, by Cytiva (1 mentions)
Anti mmp13, by Merck Group (1 mentions)
Anti ha, by Fortrea (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
3 protocols using bradford dye based assay
1
Western Blot Analysis of Protein Expression
2
Evaluating EMT Marker Modulation
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PC3 cells were treated with vehicle or with 20 μM of CUR (or DEX/CUR) for 48 h. Then proteins were extracted using RIPA buffer containing cocktail inhibitors (Sigma/Merck, Darmstadt, Germany) and concentration was determined using a Bradford dye-based assay (Bio-Rad, Milan, Italy). Total protein (30 μg of protein/lane) was subjected to SDS–PAGE followed by immunoblotting with appropriate antibodies at the recommended dilutions. The blots were then incubated with peroxidase linked secondary antibodies followed by enhanced-chemiluminescent detection using Super Signal chemiluminescence kit (Thermo Fisher Scientific, Waltham, MA, USA). The following antibodies were used: ZEB-1 (1:500; Santa Cruz Biotehcnology, Dallas, TX, USA), E-cadherin (1:1000; Cell Signaling, Leiden, The Netherlands), vimentin (1:1000; Cell Signaling, Leiden, The Netherlands), GAPDH (1:1000; Santa Cruz Biotehcnology, Dallas, TX, USA).
3
Immunoblotting Protein Expression Analysis
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Proteins were extracted with RIPA buffer containing mixture inhibitors (Roche), followed by homogenization by using QIAshredder (Qiagen) and evaluation of concentration by using a Bradford dye-based assay (Bio-Rad). 50μg of total proteins were loaded on 10% SDS/PAGE and transferred to polyvinylidene difluoride membranes (Hybond; GE Healthcare), followed by immunoblotting with appropriate antibodies. The blots were then incubated with peroxidase linked secondary antibodies followed by enhanced-chemiluminescent detection using ECL kit (Amersham). Primary antibodies were used as followed: anti-HA 1 : 100 (Covance); anti-MMP13 1:500 (Millipore); anti-actin and anti-GAPDH 1 : 10.000 (Sigma-Aldrich).
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