Western blotting was carried out as previously described with primary antibodies (dilutions) to ADAM17 (1:250), AT1 (1:500), AT2 (1:500), NICD (1:200), pERK (1:1000), pAKT (1:400), Hes1 (1:500), Jagged1 (1:500), renin (1:500), VDR (1:200), β-actin (1:3000), CK20 (1:100) and VIM (1:500) as noted in Materials (13 (link)). Proteins were measured using RC-DC assay (Bio-Rad) and lysates adjusted to the same concentration. Separate aliquots were run to blot for β-actin to confirm equal loading.
Rc dc assay
Manufactured by Bio-Rad
Sourced in United States, France
The RC/DC assay is a colorimetric protein quantification method developed by Bio-Rad. It is based on the Lowry protein assay and is designed to determine the protein concentration of samples, including those containing reducing agents or other interfering substances.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Roche (3 mentions)
Amersham hybond p membrane, by GE Healthcare (2 mentions)
P akt serine 473, by Cell Signaling Technology (2 mentions)
Phospho perk, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Thermo Fisher Scientific (2 mentions)
Gfp tag, by Proteintech (2 mentions)
Gapdh, by Proteintech (2 mentions)
β actin, by Proteintech (2 mentions)
α tubulin, by Proteintech (2 mentions)
Goat anti rabbit, by Jackson ImmunoResearch (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Protease inhibitor, by Santa Cruz Biotechnology (1 mentions)
Cpla2, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Vx 809, by Selleck Chemicals (1 mentions)
Bsa standard, by Thermo Fisher Scientific (1 mentions)
Micro bca assay, by Thermo Fisher Scientific (1 mentions)
Quant ittm picogreen dsdna kit, by Thermo Fisher Scientific (1 mentions)
Synergy htx multi mode reader, by Agilent Technologies (1 mentions)
34 protocols using rc dc assay
1
Quantitative Western Blot Analysis
2
Quantitative Analysis of Giant Ankyrin-G in Murine Hearts
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Quantitated 30 μg (RCDC assay, BioRad) 22-week murine whole-heart lysates (in SDS/BME lysis buffer (10 mM sodium phosphate buffer pH 6.8, 2 mM EDTA, 10 mM NaN3, 12 mM NaCl, 1% SDS, 1% BME)) were loaded into 3 to 8% precast gels (NuPAGE) and transferred to Tris-Acetate protein membranes, which were blocked for >1 h at room temperature in 5% milk and incubated in primary antibody overnight at 4 °C. We used the giant ankyrin-G antibody at a concentration of 1:1000 (Gift Paul Jenkins Laboratory) and Goat-anti-rabbit 1:10,000 (Jackson Laboratories) for secondary antibody (10 (link)). For control, we utilized densitometric quantification (Image Lab, Version 5.2.1 build 11.2014.) and normalized to total protein content to calculate the relative intensity of the giant AnkG isoform. For 4-week-old mice, protein expression data, 100 μg of cardiac lysates, and 10 μg of brain lysate were used to check for the expression of giant AnkG (1:750, a gift from Dr Paul Jenkins), canonical AnkG (1:1000, a gift fro m Dr Paul Jenkins), and alpha-actinin (1:1000, Millipore Sigma A7811), was used as a loading control (10 (link)). For PCR, the following AnkG primers were used: giant AnkG (480 kDa): F: 5′-GAGGCACCGCCCTTAAA-3′, R: 5′-GCCAGCTCTGTCCAACTAA-3′ and for canonical AnkG (190 kDa): F: 5′- GCTTTGCCTCCCTAGCTTTA-3′, R: 5′- GATATCCGTCCGCTCACAAG-3′.
3
Protein Extraction from Tuber Material
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Total protein was extracted from approximately 0.5 g of ground tuber material, to which 1 ml of pre-heated (95 °C) lysis buffer (50 mM sodium phosphate buffer pH 7, sucrose (5 % w/v), SDS (4 % w/v), DTT (0.3 % w/v), PVP-P (10 % w/v)) was added. Samples were homogenized and protein amount was measured using the RC/DC assay (Biorad, Veenendaal, the Netherlands).
4
Protein Purification and Analysis by SDS-PAGE and Western Blot
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Proteins were purified by precipitation in 80% (v/v) acetone at –20 °C, solubilized in protein extraction buffer [104 mM Tris–HCl pH 6.8, 3.3% SDS, 1.67× protease inhibitor (complete EDTA free, Roche), 83.3 mM DTT, 83.3 mM EDTA] and the concentration was determined with a kit (RC/DC assay, BioRad). Proteins were separated by SDS-PAGE according to Laemmli (1970) (link) on 10–15% polyacrylamide gels, and stained with Coomassie brilliant blue or blotted semi-dry onto PVDF membranes (0.2 µm pore size, Roche). The membranes were blocked for 1 h at room temperature with blocking solution [1× Roti Block (Roth) in PBS-T (137 mM NaCl, 2.7 mM KCl, 10.2 mM Na2HPO4, 2 mM NaH2PO4, 0.1% (v/v) Tween-20, pH 7.4)] and incubated in antibody solution (see Supplementary Table S3 ) for 1 h at room temperature or overnight at 4 °C. Proteins were detected with an enhanced chemiluminescence assay (ECL, GE Healthcare Life Sciences).
5
Protein Extraction and Immunoblot Analysis
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Exponential cultures with OD600 between 1-2 were lysed by vortexing with acid washed glass beads in SUMEB buffer as described previously (Xu et al. 2012 (link)). Total protein concentration was quantified using an RC/DC assay (BioRad). Equal amounts of protein were electrophoresed on 12% SDS-PAGE gels, and transferred onto Amersham Hybond-P membranes (GE). Immunoblot analysis was performed using standard procedures. All blots were scanned with a ChemiDoc XRS+ Imaging System. Band intensities were quantified using ImageJ 1.51 v software.
6
Immunoblot Analysis of Rat Brain Proteins
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The immunoblot protocol has been previously described [34 (link)]. All extracts and buffers contained protease inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA): pepstatin A (1μg/ml), leupeptin (10 μg/ml), aprotinin (20 μg/ml), phenylmethanesulfonyl fluoride (200 nM), and calpain inhibitor I and II (8 μg/ml each). Day 4 rat brain or cells grown in uncoated 60-mm dishes were solubilized in lysis buffer (0.15 M NaCl, 5 mM EDTA, 1% Triton-X 100, 10 mM Tris-Cl pH 7.4), sonicated, and centrifuged to remove insoluble material. Protein concentration was determined by the RC/DC assay (Bio-Rad, Hercules, CA). 20–30 μg of protein was loaded per lane and separated on 8% SDS-polyacrylamide gels. Equal protein loading was confirmed by staining with β-actin antibodies.
Antibodies used include: monoclonal- D2R (1:400; Santa Cruz Biotechnology), PP2B and β-actin (1:500 and 1:10,000; Sigma); polyclonal- M1R and Gqα (1:1,000; Santa Cruz Biotechnology), cPLA2 (1:200; Cell Signaling Technology, Danvers, MA), PLCβ-1 (1:200; Millipore, Billerica, MA); secondary- HRP-conjugated goat anti-mouse or bovine anti-rabbit (1:15,000 or 1:10,000; Santa Cruz Biotechnology). Primary and secondary antibodies were diluted with PBS.
Antibodies used include: monoclonal- D2R (1:400; Santa Cruz Biotechnology), PP2B and β-actin (1:500 and 1:10,000; Sigma); polyclonal- M1R and Gqα (1:1,000; Santa Cruz Biotechnology), cPLA2 (1:200; Cell Signaling Technology, Danvers, MA), PLCβ-1 (1:200; Millipore, Billerica, MA); secondary- HRP-conjugated goat anti-mouse or bovine anti-rabbit (1:15,000 or 1:10,000; Santa Cruz Biotechnology). Primary and secondary antibodies were diluted with PBS.
7
CFTR Protein Expression Analysis
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The day after transfection, cells were treated with 3 µM of VX-809 (#S1565, Selleckchem, Planegg, Germany) for 24 h before being lysed in RIPA buffer containing protease inhibitors (#04693124001, Roche Life Science, Basel, Switzerland) and protein concentration assessed using RcDc assay (#5000113, BioRad, Marnes-la-Coquette, France). Western blot analysis was performed using 60 µg of protein from each sample separated on 7% SDS-PAGE gels. After transfer onto nitrocellulose membranes, CFTR was probed using antibody 660 (Cystic Fibrosis Foundation, Chapel Hill, NC, USA) and alpha-tubulin probed with antibody DM1A (#sc32293, SantaCruz, Dallas, TX, USA). Quantification of band intensities was performed using ImageJ software (version 1.53c, NIH, Bethesda, MD, USA).
8
Protein, DNA, and VLP Quantification Assays
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Total protein concentration was determined using RC/DC assay (Bio-Rad Laboratories, Hercules, CA, USA) or Micro BCA assay (Thermo Fisher) following the manufacturer’s instructions. The calibration curve was obtained using bovine serum albumin (BSA) standards (Thermo Fisher) diluted in distilled water with a concentration range of 50–250 µg/mL. The concentration of HEK293 HCP was determined by the HEK293 host cell protein ELISA kit (Cygnus, Southport, NC, USA) according to the manufacturer’s instructions. DsDNA quantification was performed with the Quant-iTTM PicoGreen® dsDNA kit (Life Technologies). Since Gag-eGFP VLPs emit at the same range as the Quant-iTTM PicoGreen® reagent, the native fluorescence was measured prior to the reagent addition and later subtracted to the fluorescence after the reaction. Assays were performed in a 96-well plate format. The 96-well plates were read and analyzed with Synergy HTX multi-mode reader and Gen5 software, respectively (BioTek Instruments, Inc., Winooski, VT, USA).
9
Quantifying Retinal Apoptosis in Mice
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A DNA fragmentation ELISA (Cell Death Detection ELISA; Roche Applied Science, Indianapolis, IN, USA) was used to quantify total retinal apoptosis in WT and Pparα-/- mice at 4, 8, 18 and 40 weeks of age and in 11-week-old WT and Vldlr-/- mice ± fenofibric acid as described previously [16 (link), 24 (link)]. Mice were euthanized with carbon dioxide asphyxiation and retinas quickly excised. Retinas were homogenized in lysis buffer, incubated 30 minutes at room temperature, and spun down to generate a cytoplasmic fraction. The cytoplasmic fraction was then subjected to ELISA analysis as per the manufacturer’s protocol, and relative fragmentation was expressed as OD (405/490 nm) normalized by total protein content as measured by RC/DC assay (Biorad, Hercules, CA, USA) OD (750 nm). N is expressed as number of retinas per genotype, per time point.
10
Immunoblot Analysis of Muscle Oxidative Proteins
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Immunoblot was performed as previously described.47 Briefly, gastrocnemius muscle was homogenized in a buffer containing 0.23 M Tris HCl, pH 6.8, 4.5% w/v SDS, 45% glycerol, 0.04% w/v bromophenol blue, 80 mM dithiothreitol, 0.57 mM 2‐mercaptoethanol, protease inhibitor and denatured at 95°C. Concentrations were determined using the RC/DC assay (500‐0119, BioRad, Hercules, CA), and 30 μg total protein was resolved by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane and blocked in 5% weight by volume milk in Tris‐buffered saline with 0.2% Tween 20. Membranes were probed overnight for antibodies specific to CuZn Superoxide Dismutase (SOD), MnSOD, Voltage‐dependent anion channel (VDAC), Cytochrome C Oxidase Subunit IV (CoxIV), and Thioredoxin‐dependent peroxide reductase (Prdx3). Primary and secondary antibodies were diluted in Tris‐buffered saline with 0.2% Tween 20 and used according to manufacturer's protocol. Membranes were imaged using Syngene G Box. All bands were normalized to the 45 kDa actin band of Ponceau S stain as a loading control.
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