Total protein lysates were obtained using RIPA lysis buffer supplemented with 1 mM PMSF, protease inhibitor cocktail, 1 mM Na 3 VO 4 , and 10 mM NaF (Sigma Aldrich, St. Louis, MO, USA). The protein concentrations in extracts were determined by colorimetric BCA protein assays (Thermo Scientific, USA). Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on 10% Tris-glycine gels (Amresco, Solon, OH, USA) and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked for 1 h at RT with TBST (50 mM Tris-HCl, 150 mM NaCl, and 0.1% [v/v] Tween-20) containing 5% (w/v) nonfat dried milk and were subjected to immunoblotting with antibodies to CHN1 (12048-1-AP; Proteintech) and β-actin (CoWin, Beijing, China).
Colorimetric bca protein assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
Colorimetric BCA protein assays are a type of laboratory equipment used to quantify the total protein content in a sample. The assay is based on the bicinchoninic acid (BCA) method, which relies on the reduction of copper ions by proteins in an alkaline environment. The resulting purple-colored reaction product is measured spectrophotometrically to determine the protein concentration.
Automatically generated - may contain errors
4 protocols using colorimetric bca protein assay
1
Western Blot Analysis of CHN1 Protein
2
Western Blot Analysis of CHN1 Protein
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Total protein lysates were obtained using RIPA lysis buffer supplemented with 1 mM PMSF, protease inhibitor cocktail, 1 mM Na 3 VO 4 , and 10 mM NaF (Sigma Aldrich, St. Louis, MO, USA). The protein concentrations in extracts were determined by colorimetric BCA protein assays (Thermo Scientific, USA). Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on 10% Tris-glycine gels (Amresco, Solon, OH, USA) and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked for 1 h at RT with TBST (50 mM Tris-HCl, 150 mM NaCl, and 0.1% [v/v] Tween-20) containing 5% (w/v) nonfat dried milk and were subjected to immunoblotting with antibodies to CHN1 (12048-1-AP; Proteintech) and β-actin (CoWin, Beijing, China).
3
Quantifying EV Protein Content
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The EVs protein content was quantified using the colorimetric BCA protein assay (Thermo Fisher Scientific, Rockford, IL, USA). The protein concentration was measured at 562 nm, according to the manufacturer’s instructions, using a GloMax® Discover Microplate Reader.
4
Quantifying Extracellular Vesicles via BCA and NTA
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EV protein was quantified using the colorimetric BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA), per the manufacturer's instructions. Nanoparticle tracking analysis (NTA) (NanoSight NS300, Wiltshire, UK) was used to check the size distribution and the concentration of the vesicles. For NTA analysis of the EVs, PBS was used as a diluent; a syringe pump with a constant flow injection was used, and five videos of 60 s each were captured with 1498 frames and a camera level at 15. The videos were recorded and analyzsd with NTA software version 3.3 to determine the size and concentration of the particles.
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