BAL fluid (BALF) from mice or from IAV-ARDS versus control patients was concentrated using PierceTM Protein Concentrator PES, 3 K MWCO, 2–6 ml (Thermo Scientific™) and analyzed by commercial PLET1 ELISA (mouse PLET1: Antibody research corporation, USA, human PLET1: MyBioSource, USA) according to the manufacturer’s instructions. The absorbance was quantified at 450 nm wavelength in the microplate reader (iMarkTM, Bio-Rad). Human BALF protein concentration was quantified using Quick Start™ Bradford Protein Assay Kit 2 (#5000202, Bio-Rad), absorbance was quantified at 595 nm wavelength in the microplate reader (iMarkTM, Bio-Rad).
Quick start bradford protein assay kit 2
Manufactured by Bio-Rad
Sourced in United States
The Quick Start Bradford Protein Assay Kit 2 is a colorimetric assay used for the quantification of protein concentration. It is a simple and reliable method for measuring the total protein content in a sample.
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Lab products found in correlation
Pierce protein concentrator pes, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Fusion fx7, by Vilber (1 mentions)
Qproteome ffpe tissue kit, by Qiagen (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Trans blot sd semi dry transfer cell, by Bio-Rad (1 mentions)
Fusion fx7 imaging system, by Vilber (1 mentions)
Wet transfer system, by Bio-Rad (1 mentions)
Gs 800 calibrated imaging densitometer, by Bio-Rad (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Proteinext mammalian nuclear and cytoplasmic protein extraction kit, by Transgene (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Mini protean tgx stain free precast gel, by Bio-Rad (1 mentions)
Chemidoc instrument, by Bio-Rad (1 mentions)
Imagequant las 4000 mini system, by GE Healthcare (1 mentions)
Enhanced chemiluminescence reagent, by PerkinElmer (1 mentions)
7 protocols using quick start bradford protein assay kit 2
1
BALF PLET1 Quantification Protocol
2
Protein Extraction and Western Blot Analysis
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A total of 2.0×106 cells/mL were treated in six-well plates with double and triple combination regimens, and total proteins were extracted using the NE-PERW nuclear and cytoplasmic extraction kit according to the manufacturer’s protocol. Protein concentration was quantified and normalized using the Quick Start Bradford Protein Assay Kit 2 (Bio-Rad Laboratories Inc., Hercules, CA, USA) according to the manufacturer’s protocol. Fractionation was done using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose membranes. All membranes were blocked with 5% w/v BSA, 1× Tris-buffered saline and 0.1% Tween-20 at room temperature with gentle shaking for 90 minutes and incubated with primary antibodies: GAPDH (1:1000), NF-κB p65 (1:1000), IκBα (1:1000), p-IκBα (1:1000), Apaf-1 (1:1000) and caspase-9 (1:1000) overnight at 4°C, followed by detection using horseradish peroxidase-conjugated secondary antibodies (Cell Signaling), and Super Signal West Pico chemiluminescent substrate. Images were captured using the Fusion FX7 imaging system (Vilber Lourmat, Eberhardzell, Germany). Normalization of protein concentration was carried out against GAPDH as a control. Relative intensities of all bands were quantified using ImageJ v1.43 analysis software (NIH, Bethesda, MD, USA).
3
Protein Extraction and Western Blot Analysis
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Proteins were extracted from tumour biopsies using the Qproteome FFPE Tissue Kit (Qiagen, Germany) according to manufacturer's protocol. Protein concentration was quantified and normalized using the Quick Start Bradford Protein Assay Kit 2 (Bio-Rad, USA) according to manufacturer's protocol. Protein were separated on an SDS-PAGE and transferred to a 0.2 μm nitrocellulose membrane using the TransBlot-SD Semi Dry Transfer Cell (Bio-Rad, USA). Blots were blocked with 5% w/v BSA, 1×TBS, 0.1% Tween-20 at room temperature with gentle shaking for 90 min, and incubated with primary antibodies: GAPDH (1:1000), CDK4 (1:1000), MMP-9 (1:1000) overnight at 4°C. Detection of bound antibodies were done using HRP-conjugated secondary antibodies (Cell Signalling, USA), and SuperSignal West Pico chemiluminescent substrate. Images were captured using the Fusion FX7 imaging system (VilberLourmat, France). Normalization of protein concentration was carried out against GAPDH as a control. Relative intensities of all bands were quantified using ImageJ v1.43 analysis software (NIH, USA).
4
Nuclear and Cytoplasmic Protein Extraction
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Protein samples were extracted from cells (1.0 × 107/mL) after treatment using the ProteinExt®® mammalian nuclear and cytoplasmic protein extraction kit (TransGen Biotech Co., Ltd., Beijing, China). Total protein concentration was quantified and normalized using the Quick Start Bradford Protein Assay Kit 2 (Bio-Rad, Irvine, CA, USA) according to the manufacturer’s protocol. Protein samples were loaded into a sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher, Waltham, MA, USA) for Western blot analysis using a wet transfer system (Bio-Rad, Irvine, CA, USA). Then, incubation was performed with PARP 1 polyclonal antibody (1/1000) (Elabscience Biotechnology Inc., Houston, TX, USA) and beta-actin (1/2000) (Elabscience Biotechnology Inc., USA). Detection of bound antibodies was carried out using secondary HRP-conjugated goat anti-rabbit IgG (1/10,000) (Elabscience Biotechnology Inc., USA), and the bands were developed with TMB substrate (KPL, Gaithersburg, MD, USA) by viewing with a GS-800 Calibrated Imaging Densitometer (Bio-Rad, Irvine, CA, USA). The proteolytic cleavage of 116 kDa PARP into an 89 kDa fragment was assessed as the occurrence of apoptosis [47 ].
5
Bacterial Protein Extraction and Quantification
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Bacteria grown for 4 hr were collected by centrifugation 10 min at 10,000 × g at 4°C and resuspended in 500 μL of PBS. Bacteria were lysed using the Precellys lysing kit (P000914-LYSK0-A) and the Precellys program 4 (6500 rpm for 30 s) twice at 4°C. Lysates were centrifuged for 15 min at 10,000 × g at 4°C and supernatants were collected for protein quantification using the Quick start Bradford protein assay kit 2 (Biorad). Samples were mixed with Laemmli buffer (Biorad) and 10% β-mercaptoethanol and denatured for 5 min at 95°C. Samples were separated onto 4–20% Miniprotean TGX stain-free precast gel (Biorad) in TGS buffer and transferred on polyvinylidene fluoride membranes. Membranes were incubated overnight at 4°C with primary antibodies diluted in 5% blotto (R114 rabbit anti-EF-Tu polyclonal antibodies, 1:5000; R250 rabbit polyclonal anti-S. aureus NADK antibodies, 1:1000; anti-staphylococcal α-toxin (α-hemolysin) rabbit antiserum (Sigma-Aldrich S7531) 1:1000). Membranes were then incubated with 1:2500 antirabbit horseradish peroxidase-conjugated antibodies (Abcam). Blots were revealed using the ECL kit (Pierce).
6
Isolation and Purification of Mitochondria
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Cell pellets were resuspended in hypotonic buffer (4 mM Tris–HCl pH 7.8, 2.5 mM NaCl, 0.5 mM MgCl2, and 2.5 mM PMSF) and incubated for 6 min on ice. The swollen cells were disrupted by 20 strokes with a Dounce homogenizer. Isotonic levels were restored by the addition of 1/10 v/v hypertonic buffer (400 mM Tris–HCl pH 7.8, 250 mM NaCl, and 50 mM MgCl2), and pellets of nuclei and cell debris were discarded by low speed centrifugation (1,200 × g). Mitochondria were obtained by centrifugation of the remaining supernatant at 13,000 × g. Mitochondria for mitochondrial crosslinking conditions were further purified using a sucrose gradient of 1.0 M and 1.5 M. After centrifugation for 20 min at 60,000 × g, the mitochondrial layer was collected, resuspended in PBS, exposed to 302 nm UV light for 6 min in a ChemiDoc instrument (Biorad) and collected by centrifugation at 13,000 × g. Protein content of mitochondrial pellets was measured using the Quick StartTM Bradford Protein Assay Kit 2 (Biorad, 5000202) according to manufacturer’s protocol.
7
Protein Quantification and Immunoblotting Protocol
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Cells were rinsed with ice-cold PBS and lysed in RIPA buffer containing PhosSTOP and EDTA-free Protease Inhibitor Cocktail for 10 min. The soluble fractions from the cell lysates were isolated by centrifugation at 4 °C for 10 min at 13,400 × g. Next, 2×SDS buffer (4% SDS, 125 mM Tris-HCl pH 6.8, 20% glycerol, 0.01% bromophenol blue, 10% 2-mercaptoethanol) was added to the cell lysates. Protein concentrations were measured using Quick StartTM Bradford Protein Assay Kit 2(#5000202JA, Bio-Rad). Proteins were analyzed by SDS–polyacrylamide gel electrophoresis and immunoblotting following standard protocols using anti-p-JNK, anti-JNK, anti-p-Erk1/2, and anti-Erk1/2 antibodies (1:1000 dilution each). Finally, bands were visualized with the enhanced chemiluminescence reagents (Perkin Elmer, Waltham, MA) and using an ImageQuant LAS-4000 mini system (GE Healthcare, Chicago, IL). Quantification was performed with Image J software. Protein detection of p-Smad2 and Smad2 was performed with WES 004-600 (Bio-Techne, Minneapolis, MN) according to the manufacturer’s instructions.
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