Extracts were quantified with Bio-Rad Protein Assay and equal amounts of proteins were mixed in 4X SDS loading buffer before being boiled at 99 °C for 10 min. Samples were resolved onto an 8% polyacrylamide gel and transferred to 0.45 µm nitrocellulose blotting membranes (GE Healthcare). Membranes were incubated overnight at 4 °C with the primary antibodies rabbit anti-ALG-1/2 (1:1000), rabbit anti-ALG-1 (1:1000), rabbit anti-HA (1:5000) (NEB C29F4), mouse anti-V5 tag (1:500) (Abcam ab27671), mouse anti-FLAG M2 (1:5000) (Sigma-Aldrich F1804) and mouse anti β-actin (1:10,000). Membranes were then incubated with secondary antibody Sheep Anti-Mouse IgG (1:10,000) or Goat Anti-Rabbit IgG (1:5000). Imaging was done using a ChemiDoc MP imaging system (BioRad) and images were processed with Image Lab software.
Protein assay
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About the product
The Bio-Rad protein assay is a colorimetric detection and quantitation method for measuring the total protein content in a sample. It utilizes a dye-binding reagent that changes color when bound to proteins, allowing for the determination of protein concentration through spectrophotometric analysis.
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The Bio-Rad protein assay products are currently available for purchase through authorized distributors. The Bio-Rad Protein Assay Kit I and DC Protein Assay Reagent A are two examples of the company's commercialized products that can be obtained from secondary market suppliers.
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Check compatibility
7 055 protocols using «protein assay»
1
Western Blot Analysis of Protein Expression
2
Detailed Reagents for DNA Damage Assays
The following reagents were used to develop the protocols: vanadium trioxide (V2O3, CAS 1314–34–7 with 99.99 % purity), Histopaque®-1077, phosphate-buffered saline (PBS), agarose, NaCl, Nonidet P-40, DTT (1,4-dithiothreitol), sodium orthovanadate (NaVO4), sodium biphosphate (NaH2PO4), disodium phosphate (Na2HPO4), Na2-EDTA, Trizma® base, and DMSO, with all of these reagents being obtained from Sigma-Aldrich, Inc., MO, USA. The formamidopyrimidine-DNA glycosylase enzyme (Fpg) was obtained from New England BioLabs® by ChemCruz®, Dallas, USA. PB-MAXTM Karyotyping medium and RPMI-1640 medium were obtained from Gibco BRL-Invitrogen Corporation, NY, USA. Acrylamide, N,N′-methylenebisAcrylamide, glycine, sodium dodecyl sulfate (SDS), N,N,N′,N′-tetramethylethylenediamine (TEMED), tris(hydroxymethyl)aminomethane (Tris), ammonium persulfate, and the Bio-Rad protein assay were purchased from Bio-Rad Laboratories, CA, USA. The protease inhibitors aprotinin, leupeptin, and PMSF; the primary antibodies anti-ATM (sc-377293), anti-ATR (sc-515173), and anti-actin (sc-8432); the horseradish peroxidase-conjugated secondary antibody m-IgGk BP-HRP (sc-516102); the goat anti-mouse antibody IgG-HRP and Tween 20; and the Western Blotting Luminol Reagent (sc-2048) were obtained from Santa Cruz Biotechnology, Inc., CA, USA. EDTA was obtained from BD Diagnostics, Mexico. The TRIzol Reagent, RevertAid First Strand cDNA Kit (K-1622), and Maxima SYBR Green/ROX qPCR Master Mix (K-0221) were obtained from Thermo Fisher Scientific, MA, USA; additionally, Crystal Taq Master Mix (2x) was obtained from Jena Bioscience, Germany.
3
Investigating Synergistic Effects of Gem and CU17 on A549 Cells
A549 cells were seeded at a concentration of 1 × 106 cells/dish and cultured for 24 h. Cells were then treated with Gem and CU17 alone or in combination for 48 h at a synergistic condition, as previously published [9 (link)]. Thereafter, cells were initially collected with a scraper and lysed in a lysis buffer containing protease inhibitors, and the lysates were maintained on ice. A Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA) was then used to assess the concentration of the protein. Equal amounts (30–50 μg) of total protein from each treatment were resolved by SDS-PAGE and transferred to the polyvinylidene difluoride (PVDF) membrane. The membranes were blocked using a 3% nonfat milk solution for 1 h at room temperature. Afterward, PVDF membrane was incubated overnight at 4 °C with the following primary antibodies: anti-p53 (#2524, diluted 1:1000), anti-Bcl-2 (#4223, diluted 1:1000), anti-Bax (#2772, diluted 1:1000), anti-Ac-H3 (#9649, diluted 1:1000), anti-p21 (#2946, diluted 1:1000), anti-pERK1/2 (#9102, diluted 1:1000), and anti-total ERK1/2 (#9107, diluted 1:2000) (Cell Signaling, Beverly, MA, USA). The membranes were rinsed in 1X PBST for 2 min two times and then incubated with HRP-linked goat anti-mouse (#7076, diluted 1:2000) or anti-rabbit (#7074, diluted 1:2000) secondary antibodies at room temperature for 2 h following 1X PBST and PBS washes (2 × 2 min each), respectively. Ultimately, the blot was observed through the utilization of enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA, USA), and X-ray films were employed to analyze the immunoreactive bands. The measurement of relative intensity was performed using the ImageJ program, while β-actin was employed as a loading control for Western blotting to normalize the levels of protein; total ERK1/2 was employed as a loading control for pERK1/2 protein expression.
4
Biofilm Matrix Proteomics Extraction
Biofilms were grown for 24 h in TSB medium on PC chips, followed by washing once in 1x PBS to remove planktonic cells and biofilm pellicles. Five biological replicates per condition were processed, each composed of five PC chips combined to extract sufficient biofilm matrix material for proteomics analysis (technical replicates). The biofilm was then resuspended in 5 ml of 1x PBS by shaking at 1000 rpm for 10 min. The biofilm matrix was extracted using the formaldehyde and NaOH extraction method previously described [38 (link),39 ]. Briefly, 0.006 V formaldehyde 37 % (30 μl) was added to the biofilm samples and incubated for 1 h at 4 °C while mixing, followed by addition of 0.4 V NaOH 1 N (2 ml) and incubation for 3 h at 4 °C while mixing. Samples were then dialyzed using 3500 MWCO 22 mm SnakeSkin™ Dialysis Tubing (ThermoFisher Scientific) against 2.5 l Milli-Q water overnight at 4 °C with stirring. Protein content in dialyzed samples was quantified using the Bio-Rad Protein assay, a modified Bradford assay [40 (link)], before freezing with liquid nitrogen. Finally, the extracted biofilm matrix was lyophilized using an ALPHA 1–4 LDplus freeze dryer (Christ, VWR) and kept at −80 °C until sample preparation for proteomics.
For the proteomics sample preparation, digestion and mass spectrometry were conducted as described in Herschend et al. [13 ], with some modifications described in the supplementary methods.
For the proteomics sample preparation, digestion and mass spectrometry were conducted as described in Herschend et al. [13 ], with some modifications described in the supplementary methods.
5
Western Blot Analysis of Pancreatic Islet Proteins
Pancreatic islets isolated from all the experimental groups were homogenized in 80 mM Tris (pH 6.8), 5 mM EDTA, 5% sodium dodecyl sulphate (SDS), 10% glycerol, 5% dithiothreitol, and protease inhibitors (1 mM phenylmethylsulphonyl fluoride and 4 mg aprotinin). Subsequently, the samples were fractionated on SDS/PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and transferred to polyvinylidene difluoride transfer membrane (Amersham Hybond-P, GE Healthcare, Amershame, Bukinghamshire, UK) at constant 10 V for 30 min. Protein load was measured by the Bio-Rad protein assay by measuring absorbance at 595 nm. As a loading control, we used an antibody against GAPDH or β-actin, which were used as internal standards. This control was used to verify that all samples were loaded consistently on the gel and that differences in protein signals are not simply the result of variability in the amount of sample loaded and performed every run. For this reason, the results were expressed in arbitrary units (AUs) as the ratio between the protein of interest and β-actin or GAPDH band intensity. For each protein to be detected, membranes were horizontally cropped at the corresponding molecular weight. Precision Plus Protein Dual Color Standards (Bio-Rad) was used as a molecular weight marker. Cropped membranes were blocked with a 10% (w/v) non-fat milk solution for 2 h. Next, membranes were washed and incubated overnight at 4°C with specific primary antibodies [IR (CT-3-sc57342; 1:100), IRS-1 (H7-sc512070; 1:100), IRS-2 (BS-sc390761; 1:100), PI3K (D9-sc374534; 1:100), Bad (C7-sc8044; 1:100), Casp-8 ( H143-sc7890; 1:100) (sc: Santa Cruz, CA, USA), Casp-3 (C8487; 1:1000 Sigma, St. Louis, MA, USA) and 3-nitrotyrosine (621-44-3; 1:1000 Cayman, Michigan, MI, USA)]. The membranes were washed 3 times with Tween-Tris-buffered saline (T-TBS) and then incubated with the secondary antibody (anti-rabbit IgG-HRP or anti-mouse IgG-HRP) for 1 hour at room temperature. β-actin (A-1978; 1:10:000 Sigma, St. Louis, MA, USA) or GAPDH (C87727; 1:1000 Merck Millipore, Burlington, MA, USA). were used as an internal standard as appropriate. The proteins were visualized using an enhanced chemiluminescence detection system (ECL Prime, Amersham, GE Healthcare, UK). Finally, the membranes were scanned using the C-DiGit Blot scanner (LICOR), and the bands were quantified with Image Studio Digits 3.1 software. Periodically, to ensure that there are no non-specific signals generated by the binding of the secondary antibody to unrelated proteins, we performed a secondary antibody control where only the secondary antibody is applied without the primary antibody; a primary antibody control in which the primary antibody is applied in the absence of the protein of interest, confirming that the signal is not a product of nonspecific binding of the antibody; and a positive control that included a positive sample (in which the protein of interest is known to be present in high amounts) to ensure that the antibody can detect the protein of interest.
Top 5 most cited protocols using «protein assay»
1
Quantitative Analysis of Synaptic Proteins
The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology (Alexander et al., 2018 ).
Bilateral punches of cNAc and sNAc of 63 animals were sonicated using a cold buffer containing 0.32‐M sucrose, 1‐mM HEPES solution, 0.1‐mM EGTA, and 0.1‐mM PMSF, pH = 7.4, in the presence of a complete set of protease inhibitors and a phosphatase inhibitor cocktail. Total proteins have been measured in the whole homogenate by the Bio‐Rad Protein Assay (Bio‐Rad Laboratories). Western blots were run as previously described (Caffino, Giannotti, Mottarlini, Racagni, & Fumagalli,2017 ). Briefly, 10 μg of proteins for each sample was run on an 8% SDS‐PAGE under reducing conditions and then electrophoretically transferred onto nitrocellulose membranes (GE Healthcare, Milan, Italy). Blots were blocked 1 h at room temperature with I‐Block solution (Life Technologies Italia, Italy) in tris‐buffered saline + 0.1% Tween‐20 buffer and then incubated with antibodies against the total proteins of interest.
The conditions of the primary antibodies were the following:
anti‐vGlut1 (1:1000; Cell Signaling Technology Inc.; RRID:AB_2797887), anti‐GLT1 (1:5000; AbCam; RRID:AB_1566262), anti‐GluN1 (1:1000; Invitrogen; RRID:AB_2533060), anti‐GluN2B (1:1000; Santa Cruz Biotechonology; RRID:AB_670229), anti‐GluN2A (1:1000; Invitrogen; RRID:AB_2536209), anti‐SAP102 (1:1000; Cell Signaling Technology Inc.; RRID:AB_2799325), anti‐GluA1 (1:2000; Cell Signaling Technology Inc.;, RRID:AB_2732897), anti‐GluA2 (1:2000; Cell Signaling Technology Inc.; RRID:AB_10622024), anti‐SAP97 (1:1000; AbCam; RRID:AB_2091910), anti‐GRIP (1:2000; Synaptic System; RRID:AB_887728), and anti‐β‐Actin (1:10000; Sigma‐Aldrich; RRID:AB_476697).
Expression levels of every single protein was normalized using its own β‐actin loading control, which was detected by evaluating the band density at 43 kDa. Immunocomplexes were visualized by chemiluminescence using the Chemidoc MP Imaging System (Bio‐Rad Laboratories; RRID:SCCR_014210). Due to the high number of samples, they were divided across three gels that were run simultaneously and analysed. Aliquots of samples collected from SERT+/+‐naïve rats were distributed across the various gels (A–C) and used as reference values. We used the following correction factor to the different gels: correction factor gel B = average of (OD protein of interest/OD β‐actin for each sample loaded in gel A)/(OD protein of interest/OD β‐actin for the same sample loaded in gel B). Correction factor gel C = average of (OD protein of interest/OD β‐actin for each sample loaded in gel A)/(OD protein of interest/OD β‐actin for the same sample loaded in gel C). By calculating this correction factor, we were able to evaluate genotype and AMPH exposure as independent variables despite the high number of samples. Gels were run two times each, and the results represent the average from two different runs.
Bilateral punches of cNAc and sNAc of 63 animals were sonicated using a cold buffer containing 0.32‐M sucrose, 1‐mM HEPES solution, 0.1‐mM EGTA, and 0.1‐mM PMSF, pH = 7.4, in the presence of a complete set of protease inhibitors and a phosphatase inhibitor cocktail. Total proteins have been measured in the whole homogenate by the Bio‐Rad Protein Assay (Bio‐Rad Laboratories). Western blots were run as previously described (Caffino, Giannotti, Mottarlini, Racagni, & Fumagalli,
The conditions of the primary antibodies were the following:
anti‐vGlut1 (1:1000; Cell Signaling Technology Inc.; RRID:AB_2797887), anti‐GLT1 (1:5000; AbCam; RRID:AB_1566262), anti‐GluN1 (1:1000; Invitrogen; RRID:AB_2533060), anti‐GluN2B (1:1000; Santa Cruz Biotechonology; RRID:AB_670229), anti‐GluN2A (1:1000; Invitrogen; RRID:AB_2536209), anti‐SAP102 (1:1000; Cell Signaling Technology Inc.; RRID:AB_2799325), anti‐GluA1 (1:2000; Cell Signaling Technology Inc.;, RRID:AB_2732897), anti‐GluA2 (1:2000; Cell Signaling Technology Inc.; RRID:AB_10622024), anti‐SAP97 (1:1000; AbCam; RRID:AB_2091910), anti‐GRIP (1:2000; Synaptic System; RRID:AB_887728), and anti‐β‐Actin (1:10000; Sigma‐Aldrich; RRID:AB_476697).
Expression levels of every single protein was normalized using its own β‐actin loading control, which was detected by evaluating the band density at 43 kDa. Immunocomplexes were visualized by chemiluminescence using the Chemidoc MP Imaging System (Bio‐Rad Laboratories; RRID:SCCR_014210). Due to the high number of samples, they were divided across three gels that were run simultaneously and analysed. Aliquots of samples collected from SERT+/+‐naïve rats were distributed across the various gels (A–C) and used as reference values. We used the following correction factor to the different gels: correction factor gel B = average of (OD protein of interest/OD β‐actin for each sample loaded in gel A)/(OD protein of interest/OD β‐actin for the same sample loaded in gel B). Correction factor gel C = average of (OD protein of interest/OD β‐actin for each sample loaded in gel A)/(OD protein of interest/OD β‐actin for the same sample loaded in gel C). By calculating this correction factor, we were able to evaluate genotype and AMPH exposure as independent variables despite the high number of samples. Gels were run two times each, and the results represent the average from two different runs.
Corresponding organizations : University of Milan, Radboud University Medical Center, Radboud University Nijmegen, Maj Institute of Pharmacology, Polish Academy of Sciences
2
ATP Quantification in Synchronized Worms
200 age-synchronized young adult worms were collected in M9 buffer and washed three times. Worm pellets were treated with three freeze/thaw cycles and boiled for 15 min to release ATP and destroy ATPase activity, and then spun at 4°C and 11,000 g for 10 min. ATP contents were measured with a kit (Invitrogen, Carlsbad, California, USA; Cat: A22066). The ATP content value was then normalized to the soluble protein level of the same preparation, measured with the protein assay from Bio-Rad.
Corresponding organizations : McGill University
3
Whole-Cell Lysate Preparation and Immunoblotting
Whole-cell lysates were prepared in lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 0.1 mM Na2MoO4) supplemented with protease inhibitors (Complete Mini, Roche Molecular Biochemicals). Total protein concentrations were determined using the Bio-Rad Protein Assay and samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The protein-kinase array (R&D Systems, ARY003) was performed according to manufacturer guidelines. All antibodies used for immunoblotting were applied as recommended by the manufacturer. Vendor catalog numbers are provided in the Supplementary Information .
Corresponding organizations : University of Colorado Anschutz Medical Campus, Denver VA Medical Center
4
Synthesis and Characterization of [FeFe]-Hydrogenase Catalysts
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Corresponding organizations : Collège de France, Stockholm University, Laboratoire de Chimie et Biologie des Métaux, Université Grenoble Alpes, CEA Grenoble, Commissariat à l'Énergie Atomique et aux Énergies Alternatives, Centre National de la Recherche Scientifique, Max Planck Society, Ruhr University Bochum, Institut Nanosciences et Cryogénie, Université Joseph Fourier
5
Quantification and Detection of Secreted Proteins
After 48 hr, the cell culture medium was collected and centrifugated in order to remove cell debris. For protein quantification, 1 ml of cell culture medium was added with 4 ml of cold acetone, incubated for 1 hr at −20°C, and centrifuged at 14,000 g for 12 min at 4°C. The resulted pellet was resuspended in radioimmunoprecipitation assay buffer and quantified for protein concentration using Bio‐Rad Protein Assay (Bio‐Rad, Segrate, Italy). Proteins were subjected to SDS‐PAGE, followed by blotting with polyvinylidene fluoride (PVDF) membranes (Immobilon‐P transfer membrane; Millipore). PVDF membranes were incubated in 5% skimmed milk in 1× PBS for 1 hr at room temperature. After, membranes were incubated with the following primary antibodies overnight at 4°C: TGF‐β (1:250; Abcam) and IL‐10 (1:100; Santa Cruz Biotechnology). Membranes were then incubated with horseradish peroxidase (HRP)‐conjugated anti‐mouse or anti‐rat IgG secondary antibody (1:2,000; Santa Cruz Biotechnology) for 1 hr at room temperature. In order to verify the equal loading of proteins, membranes were stained with Ponceau S. Images of protein bands were visualized using an ECL system (Luminata Western HRP Substrates; Millipore), acquired by ChemiDoc MP System (Bio‐Rad) and quantified using a computer program (ImageJ; National Institutes of Health, Bethesda, MD, USA).
Corresponding organizations : Centro Neurolesi Bonino Pulejo, University of Chieti-Pescara, Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria
Spelling variants (same manufacturer)
Protein assays kit - 10 citations
Bradford reagent for protein assays - 3 citations
Bio Rad Proteins Assay - 2 citations
Bradford Proteins Assay - 2 citations
Proteins assay kit - 2 citations
Bradford protein assays kit - 2 citations
Protein Assays Kits #5000001 - 2 citations
Total cell protein assays - 2 citations
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