To evaluate the inflammatory response, the culture medium was collected from treated keratinocytes and stored at −80 °C. The protein levels of IL-22, TNF-α, IL-1β, IL-4, and IL-6 were assayed using a DuoSet ELISA kit (R&D systems) according to the manufacturer’s instructions. To evaluate the inflammatory response in the psoriasis model, the same amount of skin protein lysates from biopsies was used. The concentration of proteins was evaluated using a BCA protein assay kit (Interchim (Montluçon, France) protein quantitation kit, BCAssays, UP40840A).
Bca protein assay kit
Manufactured by Interchim
Sourced in France
The BCA protein assay kit is a colorimetric detection and quantitation method. It is used to measure total protein concentration in a sample. The assay is based on the reduction of copper ions by proteins in an alkaline medium, which results in a purple-colored reaction that can be measured spectrophotometrically.
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Lab products found in correlation
2 amino 2 methyl l propanol, by Merck Group (2 mentions)
Mgci2, by Euromedex (2 mentions)
Duoset elisa kit, by R&D Systems (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Plin4, by Novus Biologicals (1 mentions)
β actin, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Microplate reader, by Tecan (1 mentions)
Cell culture lysis buffer, by Promega (1 mentions)
Human tnfα, by Miltenyi Biotec (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Peroxidase conjugated, by Jackson ImmunoResearch (1 mentions)
Mouse gapdh, by Santa Cruz Biotechnology (1 mentions)
Rabbit histone h3, by Cell Signaling Technology (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Mouse anti psd95, by Abcam (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
19 protocols using bca protein assay kit
1
Evaluating Inflammatory Markers in Psoriasis
2
Protein Analysis of Cerebellar Tissue
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For SDS-PAGE followed by immunoblotting, protein was extracted from 25 mg cerebellar tissue of 12-week-old mice. The tissue was homogenized with a motor pestle in 10 vol. RIPA buffer [50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 % Igepal CA-630 (Sigma), 0.5 % sodium deoxycholate, 0.1 % SDS, 1 mM PMSF, Complete Protease Inhibitor Cocktail (Roche)] and incubated on ice for 15 min. After centrifugation at 4 °C and 16,000×g for 20 min, the supernatant was stored (RIPA-soluble fraction), and the remaining pellet was dissolved in ½ vol. 2 × SDS buffer [137 mM Tris–HCl (pH 6.8), 4 % SDS, 20 % glycerol, Complete Protease Inhibitor Cocktail (Roche)] by sonification followed by 10 min of centrifugation at 16,000×g. The resulting supernatant was stored as RIPA-insoluble fraction. Protein concentration was determined with the BCA protein assay kit (Interchim, France), and 20 μg of each sample were loaded onto a 7.5 % polyacrylamide gel. After gel electrophoresis, the proteins were transferred to a PVDF membrane by wet blotting. The membranes were blocked with 5 % slim milk powder in PBST and incubated with antibodies against PLIN4 (1:500, Novus Biologicals), WDFY1 (1:500, Life Span BioSciences), CNTN4 (1:1,000, Abcam), or β-ACTIN (1:10,000, Sigma). ECL (Pierce) was used for visualizing the bands, which were subsequently quantified via densitometric analysis with ImageJ.
3
Quantifying β-galactosidase Activity in Mouse Tissues
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Tissue or organotypic slices isolated from Axin2lacZ/+ mice were lysed on ice for 15 min in triton-containing lysis buffer (Applied Biosystems) and cleared by centrifugation at 10,000 g for 10 min at 4 °C. Then protein concentration was determined by BCA protein assay kit (Interchim). 50 μg of protein were applied for measuring β-galactosidase (β-gal) activity in triplicates using the “Galactostar Chemiluminescence Kit” (Applied Biosystems) in a white 96-well NUNC plate (Thermo Scientific) at room temperature (RT) for 2 h using a microplate reader (TECAN). The maximum of luminescence intensity for each individual sample was used to calculate mean of triplicate measurements. Lysates of corresponding β-gal-negative brain tissues were used to calculate background intensity.
4
Betaine and C-PC Modulate NF-κB Pathway
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A549/NF-κB-Luc cells treated for 24 h with betaine and/or C-PC were washed three times with PBS (Sigma-Aldrich), and DMEM without FBS was added along with human TNFα (Miltenyi Biotec) at final concentration of 25 ng·mL−1 as an inductor of the NF-κB pathway. Following 9 h incubation, cells were washed once in PBS, trypsinised, mixed with complete medium, and centrifuged for 7 min at 1500 ×g and the dried pellet was stored at −80°C until the day of analysis. The samples were thawed and washed with 500 μL PBS (5 min, 1500 ×g). The pellet was resuspended in 300 μL of Cell Culture Lysis buffer (Promega) with TritonX-100, vortexed for 30 s, and incubated for 10 min at room temperature. Samples were centrifuged for 10 min at 3000 ×g, 60 μL of supernatant was put in the luminometer tubes on ice, and 100 μL mixture of lysis buffer with 10 mM ATP was added to each tube. Luciferase activity was measured using a luminometer (LUMAT LB 9507), normalized to total cell protein using a BCA protein assay kit (protein quantification kit—BC assays, Interchim, Montluçon Cedex, France) and expressed as relative light units (RLU) per mg protein.
5
Fecal Protein Extraction and Lysozyme Activity
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Faecal proteins were mechanically extracted after rehydration of frozen faeces in 500 µl of PBS (10 mM; pH 7.2). After 10 minutes of centrifugation at 1600 g, supernatants were sterilized with a 0.22 µm filter and frozen (−80 °C). Faecal protein concentration was measured using BCA protein Assay kit, Uptima (Interchim, Montluçon, France). Activity of lysozyme against the peptidoglycan was determined using the EnzChek® Lysozyme Assay Kit (Molecular probes, life technology, St Aubin, France).
6
Quantifying Calcium Content in HASMCs
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The calcium contents in the HASMCs and aortas were quantified using a calcium detection assay kit (ab10205, Abcam, Cambridge, MA, USA). Sample preparation and assay procedures were performed in accordance with the instructions provided in the kit. The calcium contents were normalized to the protein concentrations, which were determined using a BCA protein assay kit (Interchim, Montluçon, France).
7
Quantification of Protein Expression
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Cells were harvested on ice, washed with cold PBS, and collected on RIPA buffer Biobasic supplemented with protease inhibitor (Sigma). Protein concentration was measured using BCA Protein Assay Kit (Interchim), and equal amounts of proteins were subjected to SDS-PAGE (TGX Stin Free FastCast Acrylamid, 12%, Bio-Rad, 161–0185) and transferred onto nitrocellulose membrane (Transblot Turbo, Bio-Rad, 1704271). Membranes were washed in Tris-buffered saline supplemented with 0.05% Tween-20 and then saturated in Tris-buffered saline supplemented with 0.05% Tween-20 with 5% BSA, incubated overnight with primary antibodies in Tris-buffered saline supplemented with 0.05% Tween-20 with 5% BSA, washed and revealed with Clarity Western ECL Substrate (Bio-Rad, 170–5060). Western blotting was conducted using standard methods with the following antibodies: Rabbit anti-PSPC1 (bethyl laboratory, A303-205A) diluted 1:1000, Rabbit anti-P54nrb (Santacruz, sc67016) diluted 1/400, Rabbit Histone H3 (Cell Signaling, 4499) diluted 1/10000, mouse GAPDH (SantaCruz, SC32233) diluted 1/1000, secondary donkey anti-rabbit IgG antibody, Peroxidase Conjugated, (Jackson ImmunoResearch, 711-035-152) diluted 1:10000, secondary rabbit anti-mouse IgG antibody, Peroxidase Conjugated, (Jackson ImmunoResearch, 715-035-150) diluted 1:10000.
8
ALP Activity Quantification in Cell Lysates
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At D3, cells seeded in 24-well plates were rinsed in PBS, lysed in deionized water and stored at −80°C. Lysates were then sonicated and centrifuged at 10,000 rpm for 5 min. To measure the ALP activity, 20 µl of the supernatants of lysates were added to 180 µl of a mixture (0.1 M 2-amino-2-methyl-l-propanol (Sigma-Alrich, St Quentin-Fallavier, France), 1 mM MgCI2, 9 mM p-nitrophenyl phosphate (pNPP) (Euromedex, Mundolsheim, France), pH 10) in 96-well plates. The ALP activity was assayed by measuring the absorbance at 405 nm using a Multiskan EX plate reader (Labsystem, Helsinki, Finland). Total protein amounts of each lysate were quantified using a BCA protein assay kit (Interchim, Montluçon, France). The activity was expressed as a percentage relative to the activity of the positive control.
9
ALP Activity Assay for C2C12 Cells
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After 3 d in culture on BMP-2 loaded films in 96-well plates, C2C12 cells were assayed for ALP activity, a marker for osteoblast differentiation. After removal of culture medium, cells were lysed by adding a 50 µl of 0.5% Triton-X100 in PBS supplemented with 0.15 µl benzonase (Novagen) and 1× antiprotease (antiprotease complete; Roche). The plate was put at 37°C for 20 min, and half of the volume was conserved for the protein assay. A buffer containing 0.1 M 2-amino-2-methyl-l-propanol (Sigma-Aldrich), 1 mM MgCI2, and 9 mM p-nitrophenol phosphate (Euromedex), adjusted to pH 10.0 with HCl, was used to assay the cell lysate for ALP. Reaction was followed over 5 min in a 96-well plate by measuring the absorbance at 405 nm using a Tecan Infinite 1000 Microplate reader. The activity was expressed as µmoles of p-nitrophenol produced per minute per milligram of protein. Total protein contents of the samples were determined using a BCA protein assay kit (Interchim).
10
Protein Expression Analysis in EAE Mice
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Hippocampal tissue isolated from EAE mice was lysed on ice for 15 min in RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1 % (v/v) NP40, 0.5 % (v/v) sodium deoxycholate, 0.1 % (v/v) SDS) with protease and phosphatase inhibitors (Roche). Lysates were cleared by centrifugation at 14,000 g for 20 min at 4 °C and protein concentration was determined using a BCA protein assay Kit (Interchim). Protein samples were loaded on 8–16 % gradient “Mini-Protean TGX” gel (Biorad). Blotting onto a nitrocellulose membrane was performed using the “Trans-Blot Turbo System” (Biorad) for 7 min at 25 Volts. Afterwards, membranes were incubated with blocking buffer (5 % (v/v) skimmed milk in 0.05 % (v/v) PBS/Tween) for 1 h at RT and incubated overnight at 4 °C with following antibodies: mouse anti-neurofilament 160/200 (Sigma-Aldrich), mouse anti-PSD-95 (Abcam), rabbit anti-β-catenin (Cell Signalling), mouse anti-active-β-catenin (ABC, Cell Signalling), mouse anti-β-actin (Sigma-Aldrich). Primary antibodies were detected by incubation with IR-Dye secondary antibodies (Li-COR) for 1 h at RT and subsequently quantified using the Odyssey infrared imaging system (LI-COR). Optical density analysis was performed with ImageJ software (NIH).
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