The brain tissue from all experimental groups was homogenized in ice-cold RIPA buffer containing, PMSF (1 mM) and protease inhibitor cocktail (1 μl/ml of lysis buffer; Sigma Aldrich, St. Louis, MO, USA), then after the protein was extracted. The extract was centrifuged at 12,000×g for 15 min at 4 °C. The supernatant was collected, aliquoted, and stored at −80 °C until further use. Protein content in the different samples was measured with Bradford dye (Bio-Rad) in a 96-well microtiter plate against a bovine serum albumin (BSA) standard. The plate was read at 594 nm in a Spectra Max M2 plate reader (Molecular Device).
Bradford dye
Manufactured by Bio-Rad
Sourced in United States, Canada
The Bradford dye is a colorimetric assay for protein quantification. It utilizes the binding of Coomassie Brilliant Blue G-250 dye to proteins, resulting in a color change that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (7 mentions)
Spectramax m2, by Molecular Devices (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Spectramax m2 plate reader, by Molecular Devices (2 mentions)
Ecl solution, by Thermo Fisher Scientific (2 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Tris base t9200, by GenDEPOT (2 mentions)
Anti acta1 mab1501, by Merck Group (2 mentions)
Gapdh, by Abcam (1 mentions)
Ab1416, by Abcam (1 mentions)
E cadherin, by Abcam (1 mentions)
Vimentin ab92547, by Abcam (1 mentions)
β catenin ab32572, by Abcam (1 mentions)
α sma, by Cell Signaling Technology (1 mentions)
Phosstop, by Roche (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Phospho src, by Cell Signaling Technology (1 mentions)
33 protocols using bradford dye
1
Protein Extraction from Brain Tissue
2
Western Blot Quantification of EMT Markers
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After treatment, total proteins were extracted from cells using RIPA lysis buffer containing protease inhibitors. Protein concentrations were measured by Bradford dye (Bio-Rad). 40 μg proteins were loading to the SDS-PAGE gel, and then transferred to PVDF membranes (Millipore, Billerica, MA, U.S.A.). Then, the PVDF membranes were blocked by 5% milk and further immersed in primary antibodies overnight at 4°C. After washing with TBST, the membranes were incubated with the secondary antibody (Cell Signaling, U.S.A.) (1:5000 dilution) for 1 h at RT. After TBST wash, ECL solutions (Thermo Pierce) were used to detect the protein bands. GAPDH was served as an endogenous control. Antibodies used include E-cadherin (ab1416, 1:2000 dilution), Vimentin (ab92547, 1:2000 dilution), β-catenin (ab32572, 1:2000 dilution), and GAPDH (ab8245, 1:3000 dilution) were purchased from Abcam (U.S.A.). α-SMA was purchased from Cell Signaling (Cell Signaling 19245, 1:2000 dilution).
3
Platelet-Dependent Monocyte Signaling Assay
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Cell lysates were prepared by incubating platelet containing or platelet-deprived monocytes in 1% Triton X-100 in 10 mM tris (pH 7.4) and 150 mM sodium chloride + protease inhibitors (cOmplete Protease Inhibitor Cocktail, Roche) + phosphatase inhibitors (PhosSTOP, Roche) at 4°C for 1 hour. Protein estimation of the clarified lysate was carried out using Bradford dye (Bio-Rad). Laemmli sample buffer was added to clarified lysates containing equal amount of protein and subjected to SDS–polyacrylamide gel electrophoresis, followed by Western blotting. Antibodies used for analysis, c-Src, ATK, p-AKT, GRP94, glyceraldehyde-3-phosphate dehydrogenase (Proteintech), Phospho-Src, ERK, Phospho-ERK (Cell Signaling Technologies), anti-MART1 antibody (A103; a gift of R. Halaban), or anti-OVA monoclonal antibody (mAb) (Rockland), were used as per the supplier’s recommendations.
4
Western Blot Protein Detection
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Total protein was isolated using RIPA lysis buffer containing protease inhibitors. Protein concentrations were measured by Bradford dye (Bio-Rad). Protein (50μg of each sample) were separated by 10% SDS-PAGE and transferred electrophoretically onto PVDF membranes (Millipore, Billerica, MA, USA). Then, the membranes were blocked in 5% skim milk for 1 h, washed three or four times with Tris-buffered saline containing 20% Tween 20 (TBST) at room temperature, and incubated with the primary antibodies at 4°C overnight. Following washing with TBST, the membranes were then immersed in secondary antibody (goat anti-rabbit IgG, 1:1000, Santa Cruz Biotechnology) at room temperature for 1 h. After TBST wash, the immunoreactivity was visualized by ECL solutions (Thermo Pierce). β-actin (Santa Cruz Biotechnology) served as an endogenous control.
5
Purification of Histidine-Tagged Proteins
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Transformed cells were resuspended in 40 mL of iced cold lysis buffer (50 mM Tris HCl at pH 8.0, 0.15 M NaCl) on ice. Cells were lysed on ice by sonication at a 30% amp output for 3 min (20 sec pulses) and lysate was centrifuged at 4000×g for 20 min at 4°C. Lysate was purified by affinity chromatography. Soluble protein lysate was added to Ni-NTA resin (ThermoScientific) and incubated for 2 h at 4°C. The resin was centrifuged at 800×g for 5 min at 4°C and the resin was washed 10×1.5 mL with wash buffer (50 mM Tris HCl at pH 8.0, 0.15 mM NaCl, 20 mM imidazole). Protein was eluted off of the resin with 50 mM Tris HCl at pH 8.0, 0.15 mM NaCl, 1 M imidazole. Eluted protein was monitored by Bradford dye (BioRad), pooled, and exposed to final concentrations of 1 mM DTT, 1 mM EDTA, and 0.5 M ammonium sulfate. Protein was incubated for 1 h at 4°C before extensive dialysis against 25 mM HEPES at pH 7.4, 10% glycerol, 0.01% Triton X-100. Dialyzed protein was centrifuged at 2000×g to remove any precipitated protein. Purity was determined by SDS-PAGE and monomer concentration was determined by UV-Vis at 280 nm using a molar extinction coefficient of 14,800 M−1cm−1.
6
Western Blot Analysis of Apoptotic Proteins
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Whole-cell lysates were prepared from BGC823 cells, and standard Western blot analysis was performed. Protein concentrations were detected using Bradford dye (Bio-Rad Laboratories Inc., Hercules, CA, USA). Equal amounts of protein extracts (10 μg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membrane was probed with specific primary antibodies including XIAP (1:300), cyclin D1 (1:300), BCl-2 (1:800), Bax (1:600), and caspase-3 (1:600). The protein bands were normalized with β-actin, and all the blots were quantified with Software Quantity One (Bio-Rad Laboratories Inc.).
7
Ubiquitin-Protease Assay and Reagents
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All chemicals and reagents were from Sigma Aldrich unless otherwise stated. Solvents (except DMSO) were from Fisher (Pittsburg, PA). Other reagents used in this study: PEITC (Acros Organics); Bortezomib (Millennium Pharmaceuticals); Mini-Complete and PhosSTOP inhibitory cocktails (Roche Applied Science); recombinant human K63-linked di-ubiquitin (UC-300), recombinant human His6-USP1/UAF1 complex (E-568; Boston Biochem.); DMEM, glutamax, penicillin/streptomycin (Gibco); trypsin (0.25%), DPBS (Hyclone); cell dissociation buffer (fisher); Bradford dye (Bio-rad); dithiothreitol (GoldBio Tech); PVDF hybond, Amersham ECL Prime WB detection reagent (GE Healthcare Life Sciences); Blue Biofilm (Denville Scientific); ML323 USP1 inhibitor (EMD Millipore); TransIT 2020 transfection reagent (Mirus bio); Pierce protein G magnetic beads (ThermoFisher Scientific); TAMRA-ubiquitin propargylamide (TAMRA-Ub-PA), Cy5-ubiquitin vinyl methyl ester (Cy5-Ub-VME), Biotin-Ahx-Ub-VME, Biotin-Ahx-Ub-PA and Ub-Rh110MP (UbiQ). HA-ubiquitin vinylsulfone (HA-Ub-VS) and HA-Ub-VME were synthesized using standard methods previously described [88 (link)]. The plasmid encoding the HA-Ub(1-75)-intein-chitin binding domain fusion protein was a gift from Prof H. Ploegh of the Whitehead Institute.
8
Total Protein Extraction and Immunoblotting
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For total protein extraction, cells were washed with cold PBS and resuspended in TOTEX buffer [20 mM Hepes (pH 7.9), 350 mM NaCl, 20% (v/v) glycerol, 1% (v/v) NP-40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 10 mM NaF, 0.5 mM sodium orthovanadate, and protease inhibitors]. Protein concentration was quantified with Bradford Dye (Bio-Rad), and 20 to 30 μg of protein were resolved with SDS–polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad) and blocked with 5% nonfat milk in PBST (PBS with Tween 20) for 1 hour at room temperature. Primary antibodies (see table S2) were incubated overnight in a cold room on a rotating platform. Membranes were washed with PBST six times, 10 min each. Horseradish peroxidase–conjugated secondary antibodies (see table S3) were incubated at 1:5000 dilution for 1 hour at room temperature. Membranes were washed with PBST six times, 10 min each, and enhanced chemiluminesence reagent was incubated with membrane for 1 min before images were developed in a dark room.
9
Histone Protein Extraction from Cells
10
Muscle Tissue Protein Extraction
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