Whole pasteurized milk from cow, goat and sheep were purchased in local supermarkets (Madrid, ES). PierceTM BCA Protein Assay Kit and PierceTM Detergent Compatible Bradford Assay Kit were purchased from ThermoFisher Scientific (Rockford, IL). SDS and BluSafe dye were acquired from Sigma-Aldrich (St. Louis, MO) and Nzytech (Lisbon, PT), respectively. CriterionTM XT precast gels with 12% Bis-Tris, sample buffer, Precision Prestained Protein Dual Xtra Standard (250 kDa to 2kDa) and Bis-Tris SDS running buffer were obtained from Bio-Rad Laboratories (Hercules, CA).
Pierce detergent compatible bradford assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce Detergent Compatible Bradford Assay Kit is a colorimetric assay used for the quantitation of total protein. It is designed to work with samples containing detergents, which can interfere with other protein assays.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Mini protein tgx gel, by Bio-Rad (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Fluostar optima, by BMG Labtech (2 mentions)
Mini gel tank, by Thermo Fisher Scientific (2 mentions)
Hrp labeled secondary antibody, by Thermo Fisher Scientific (2 mentions)
Iblot 2 dry blotting system, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bolt 10 bis tris plus gel, by Thermo Fisher Scientific (2 mentions)
Cd177, by Abcam (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Seahorse xfe96 extracellular flux analyser, by Agilent Technologies (1 mentions)
Y per reagent, by Thermo Fisher Scientific (1 mentions)
Pvdf transfer membrane, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus kit, by Thermo Fisher Scientific (1 mentions)
Complete ultra protease inhibitor, by Roche (1 mentions)
Tris acetate sds running buffer, by Thermo Fisher Scientific (1 mentions)
34 protocols using pierce detergent compatible bradford assay kit
1
Milk Protein Quantification Methods
2
Protein Quantification and Western Blot
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The tissues were lysed, and total protein was quantified using the PierceTM Detergent Compatible Bradford Assay Kit (Thermo Scientific). 20 μg of protein from each sample was used for SDS-PAGE. After transferring the sample onto a PVDF membrane, the blot was incubated with indicate antibodies. All antibodies were purchased from CST: CD177 (ab203025, Abcam), TTPAL (ab103740, Abcam), FLT3 (#3462, CST), and GAPDH (#5174, CST).
3
Western Blot Analysis of Inflammatory Markers
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The stomach tissue from each animal was homogenized using the bullet blender homogenization kit (Advance Inc., Newark, DE, USA). Tissue extracts were centrifuged at 13,000 rpm for 20 min at 4°C to remove insoluble materials. The total concentrations of extracted protein were determined using Bradford assays (PierceTM Detergent Compatible Bradford Assay Kit, Thermo Fisher Scientific, Waltham, MA, USA). Aliquots of each protein sample (50 μg) were separated on 8% sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. After blocking for 10 min with Tris-HCl, 150 mM NaCl and 0.05% Tween-20 (TBST) (pH 7.6) containing 5% skim milk for 1 h at room temperature (RT), the membranes were washed with TBST. Membranes were then incubated with diluted primary antibodies, including anti-iNOS and anti-COX-2, overnight at 4°C. The blots were subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Inc.) for 1 h at RT. Blots were washed three times with TBST buffered saline, and the immunoreactive protein bands were visualized with enhanced chemiluminescence (Thermo Fisher Scientific).
4
Protein Extraction and Quantification
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Tissues were isolated for protein by using RIPA Lysis and Extraction Buffer (Thermo Scientific) containing 1× Pierce™ Protease Inhibitor Tablet, EDTA-free (Thermo Scientific) following the manufacturer’s instructions. Protein samples were quantified using the Pierce™ Detergent Compatible Bradford Assay Kit (ThermoFisher) (Bradford, 1976 (link)) before Western blot analysis.
5
Western Blot Analysis of NMP4 Protein
6
Mitochondrial Function Assessment in Cells
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A total of 5 × 103 cells per well were seeded into Agilent XFe96 Seahorse plates, transduced 24 h later with pAAV-ophNdi1 or pAAV-Ndi1, at an MOI of 3.4 × 105. A mitochondrial stress test was performed 48 h later according to the manufacturer’s protocols (Seahorse XFe96 extracellular flux analyser, Agilent Technologies, CA, USA). Injection cycles were 5× for basal OCR, 5× following oligomycin (1 µM), 5× following FCCP (1 µM), 5× following rotenone (0.5 µM) and 5× following antimycin A (0.5 µM) injections [4 (link),49 (link)]. Six replicas per group were analysed. Basal and maximal OCRs, SRC and OCR rescue post-rotenone treatment (measurement 16–20/measurement 11–15 × 100) were determined. Primary fibroblasts were analysed as described above, but with 2.25 µM FCCP. The ATP Rate Assay was carried out as per the manufacturer’s protocol (Agilent Technologies, CA, USA) using 5 × 103 cells per well. Seahorse analyses were normalised using the Pierce™ detergent compatible Bradford assay kit (Thermo Fischer Scientific, MA, USA), with absorbance determined at 595 nm (FLUOstar OPTIMA; BMG Labtech, Aylesbury, UK) and a standard test sample included on all Seahorse plates in a given experiment.
7
Furfural and HMF Reducing Activities
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For the analysis of furfural and HMF reducing activities, the K. marxianus strains S9 and CBS6556 were grown for 24 h in YPD medium at 37 °C and 200 rpm in an orbital shaker. The cells were collected, and the crude protein extracts were prepared with Y-PER reagent (Thermo Fisher Scientific, Waltham, MA, USA) The total protein concentration was determined using the Pierce™ Detergent Compatible Bradford Assay Kit (Thermo Fisher Scientific).
Furfural and HMF-reducing activities were assayed in each of the cell extracts (in triplicate) by measuring the decrease of NAD(P)H at 340 nm and 30 °C using a microplate reader spectrophotometer. The reaction mixtures were prepared as described by Nilsson et al. (2005) [24 (link)]: 100 mM phosphate buffer pH 7.0, 10 mM furfural or HMF, 100 µM of NADH or NADPH, and 20 µL of crude extract (properly diluted). Furfural and HMF reducing activities are expressed as units per mg of protein (U/mg protein), in which units (U) is defined as μmol of NAD(P)H oxidized per min.
Furfural and HMF-reducing activities were assayed in each of the cell extracts (in triplicate) by measuring the decrease of NAD(P)H at 340 nm and 30 °C using a microplate reader spectrophotometer. The reaction mixtures were prepared as described by Nilsson et al. (2005) [24 (link)]: 100 mM phosphate buffer pH 7.0, 10 mM furfural or HMF, 100 µM of NADH or NADPH, and 20 µL of crude extract (properly diluted). Furfural and HMF reducing activities are expressed as units per mg of protein (U/mg protein), in which units (U) is defined as μmol of NAD(P)H oxidized per min.
8
Protein Isolation and Western Blot Analysis
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Proteins were isolated from two to three human cortical organoids at 6 weeks or from N2a cells that overexpressed human EREG for 24 h. Briefly, N2a cells were plated in six-well plates and grown in N2a medium containing DMEM/F12, 10% FBS, and 1X penicillin/streptomycin. Cells were transfected with a pCAG-empty or pCAG-EREG plasmid, together with pCAG-GFP plasmid, using lipofectamine 2000 according to the manufacturer’s instructions. Protein concentration was measured using the Pierce detergent-compatible Bradford assay kit (Thermo Scientific, #23246). Subsequently, 40 µg of protein were resolved on a 10% SDS Polyacrylamide gel and transferred to a PVDF transfer membrane (Thermo Scientific, #88518). Membranes were blocked for 1 h at room temperature with 5% skim milk in PBS with 0.05% tween, and then incubated with primary antibodies overnight at 4 °C. Secondary antibodies were incubated for 1 h at room temperature. Antibody signal was detected using the SuperSignal West Pico plus kit (Thermo Fisher Scientific, #34579).
9
Protein Isolation and Separation
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Total protein was isolated from cells using RIPA lysis buffer [50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, cOmplete ULTRA protease inhibitors (Roche #05–892-791-001)]. Harvested cells were washed 2 times in cold PBS, subsequently resuspended and incubated in cold RIPA lysis buffer for 20 min on ice. Samples were sonicated using a Diagenode Bioruptor (5 cycles of 30 seconds on/off at high power) and cleared by centrifugation at 18 000 g for 15 min at 4°C. Protein concentration in the cleared supernatant was determined using the Pierce Detergent Compatible Bradford Assay Kit (Thermo Fisher #23246) according to the manufacturer's instructions. Subsequently, 15 μg of total protein per well was loaded into Novex NuPAGE Tris-Acetate 3–8% polyacrylamide gels (Thermo Fisher #EA03785BOX) in Tris-Acetate SDS running buffer (Thermo Fisher #LA0041) and the protein bands were separated according to manufacturer's instructions. Bands from the gel were transferred to 0.45 μm PVDF membrane (MilliporeSigma #IPFL00010) overnight.
10
Quantification of Perilipin-2 Protein Levels
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Briefly, 10 guts per sample were lysed in 2 X SDS-loading buffer and protein was quantified using Pierce Detergent compatible Bradford Assay kit (Thermo Scientific). The lysates were separated by SDS-PAGE and transferred to Nitrocellulose membrane. The membrane was blocked with 5% non-fat dry milk in TBST and incubated with primary antibodies against Perilipin-2 (1:4000 gift from Dr. Michael Welte) [13 (link),46 (link)] and Actin (1:1000, Cell Signaling). HRP conjugated Secondary antibodies were used for detection using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA). The western blot images were captured by a BIO-RAD workstation.
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