The protease activity of the culture supernatant on casein was detected using the Folin phenol method [10 (link)]. One unit of protease activity was determined as the amount of enzyme that catalyzed the formation of 1 µg tyrosine per min. The protein contained in the supernatant was quantified using the BCA protein quantification kit (Biosharp, Anhui, China), and bovine serum albumin (Biosharp, Anhui, China) at concentrations of 0, 0.2, 0.4, 0.6 and 1 mg/mL was used as the standard for the determination of the protein content [28 (link)].
Bca protein quantification kit
Manufactured by Biosharp
Sourced in China
The BCA protein quantification kit is a colorimetric assay used to determine the concentration of proteins in a sample. The kit utilizes the bicinchoninic acid (BCA) reaction, where proteins reduce copper ions to a purple-colored complex that can be measured spectrophotometrically. The kit provides a simple and reliable method for quantifying total protein content in a wide range of sample types.
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Lab products found in correlation
Protease inhibitor cocktail, by Takara Bio (2 mentions)
Excess streptavidin magnetic beads, by Yeasen (2 mentions)
Sds page loading buffer, by Takara Bio (2 mentions)
Ripa buffer, by Biosharp (2 mentions)
Bovine serum albumin (bsa), by Biosharp (1 mentions)
Anti stat3, by Proteintech (1 mentions)
Anti pstat3, by Affinity Biosciences (1 mentions)
Anti jak1, by Proteintech (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Total exosome isolation reagent, by Thermo Fisher Scientific (1 mentions)
H 600, by Hitachi (1 mentions)
5 protocols using bca protein quantification kit
1
Quantifying Protease Activity and Protein Content
2
Metabolic Labeling and Enrichment of Proteins
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l -AHA-labeled cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Biosharp) containing protease inhibitor cocktail (TaKaRa). Proteins from cell lysis were collected and quantified using the bicinchoninic acid (BCA) protein quantification kit (Biosharp), and the protein concentration was adjusted. By clicking chemical reactions, proteins containing l -AHA were labeled using BPDO-PEG4000-biotin (MCE) with a final concentration of 40 μM. Excess streptavidin magnetic beads (YEASEN) were added to the protein mixture for adsorption of biotin-labeled proteins, and the unbound proteins were washed away with a RIPA buffer. The remaining beads and proteins were added with 40 μl 4× SDS-PAGE loading buffer (TaKaRa) containing 2 mM BPDO-PEG4000-biotin and incubated at 98°C for 10 min. Samples were stored at –80°C for subsequent detection.
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3
Metabolic Labeling and Enrichment of Proteins
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l -AHA-labeled cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Biosharp) containing protease inhibitor cocktail (TaKaRa). Proteins from cell lysis were collected and quantified using the bicinchoninic acid (BCA) protein quantification kit (Biosharp), and the protein concentration was adjusted. By clicking chemical reactions, proteins containing l -AHA were labeled using BPDO-PEG4000-biotin (MCE) with a final concentration of 40 μM. Excess streptavidin magnetic beads (YEASEN) were added to the protein mixture for adsorption of biotin-labeled proteins, and the unbound proteins were washed away with a RIPA buffer. The remaining beads and proteins were added with 40 μl 4× SDS-PAGE loading buffer (TaKaRa) containing 2 mM BPDO-PEG4000-biotin and incubated at 98°C for 10 min. Samples were stored at –80°C for subsequent detection.
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4
Western Blot Analysis of JAK/STAT Pathway
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We extracted total protein from cells using RIPA lysis buffer. Protein quantification was performed using BCA Protein Quantification Kit (Biosharp, Hefei, China) according to the protocol. Sample protein (25 μg) were loaded, followed by SDS-PAGE electrophoresis. Then the purpose tape was transferred to polyvinylidene fluoride (PVDF) membranes. After rinsed with TBST for 5 min (3 times), the membranes were blocked with 5% skim milk for 2 h at 37°C. After washing by TBST for three time, membranes were immersed in the primary antibodies incubation solution (anti-JAK1; dilution 1:1000; Proteintech, Chicago, IL, USA); anti-STAT3 (dilution 1:1000; Proteintech); anti-p-STAT3 (dilution 1:1000; Affinity, Jiangsu, China); anti-GAPDH (dilution 1:5000; Proteintech) overnight at 4°C. The secondary antibodies (goat anti-rabbit or goat anti-mouse HRP-conjugated IgG; dilution 1:5000; ZSGB-BIO, Beijing, China) were added with the membranes at 37°C for 1 h. ECL luminescent solution was added to the membrane, and images were taken using Integrated chemiluminescence instrument. Quantitative analysis was performed using Image J software.
5
Exosome Isolation and Characterization
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When cell confluency reached 80%, serum-free medium was replaced. After 72 h of culture, the cell culture supernatant was collected, and Exo was extracted using Total Exosome Isolation Reagent (Cat#4478359, Invitrogen, USA). BCA protein quantification kit (Cat# BL521A, Biosharp Co. Ltd., Hefei, China) was used to detect Exo protein content. The morphology of Exo was observed under a transmission electron microscope (HITACHI, H-600). Western blot was used to identify the expression of Exo-specific markers CD9 (ab19761, Abeam) and heat shock protein (HSP) 70 (bs-0126R, Bioss).
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