MDBK were inoculated with BoHV−1, BoHV−1 ΔgE, BoHV−1 gE/E2−Linker−E2+, and BVDV−1 (cp). Cell samples were collected when the cytopathic effect reached about 90%, and sample concentration was determined with a BCA protein concentration detection kit (Thermo, TK274303, Waltham, MA, USA). Proteins were separated by SDS polyacrylamide gel electrophoresis and transferred to a PVDF membrane. A total of 5% skimmed milk was blocked at room temperature for 2 h. The PVDF membrane was washed three times with TBST, BVDV−1 E2 specific mAb (1:1000 dilution) was incubated at 4 °C for 10 h, and the PVDF membrane was washed three times with TBST. Goat Anti-Mouse IgG H&L (HRP) (1:5000 dilution) was incubated at room temperature for 1 h and the PVDF membrane was washed three times with TBST. The PVDF membrane was observed using ECL chromogenic solution (Thermo, SK255593) in a protein-scanning imager.
Bca protein concentration detection kit
Manufactured by Thermo Fisher Scientific
Sourced in China, United States
The BCA protein concentration detection kit is a colorimetric assay used to determine the total protein concentration in a sample. The kit utilizes the bicinchoninic acid (BCA) reagent, which reacts with proteins to produce a purple-colored complex that is measured spectrophotometrically. The intensity of the color is proportional to the protein concentration in the sample.
Automatically generated - may contain errors
Lab products found in correlation
0.22um lter, by Merck Group (3 mentions)
Hyaluronidase and polyethylene glycol peg6000, by Merck Group (3 mentions)
H 7650 transmission electron microscope, by Hitachi (3 mentions)
Cryostat centrifuge, by Thermo Fisher Scientific (3 mentions)
Proteinase inhibitor cocktail, by Merck Group (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
6 protocols using bca protein concentration detection kit
1
BoHV-1 and BVDV-1 Protein Analysis
2
Protein Extraction and Quantification
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Cells were lysed using RIPA lysis buffer (Solarbio, Beijing, China) with proteinase inhibitor cocktail (Sigma, St. Louis, MO, USA) and phosphorylase inhibitor cocktail II and III (Sigma). The concentration of protein lysates was detected by BCA protein concentration detection kit (Thermo Fisher, San Francisco, CA, USA). A total of 30 μg protein is used at each Western blot. The protein was loaded to vertical electrophoresis and transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with primary antibody and subsequent secondary antibody. The bands were detected by a Tanon ECL imaging system. ImageJ software was used to quantify the immunoblots. Area, mean gray value, and integrated density of immunoblots were measured during the process. For detailed description and the antibodies used this process, see Table S4.
3
Exosomal Protein Profiling Workflow
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The protein expression of CD63, TSG101 and HSP70 in the exosomes from the plasma of patients or culture supernatant was measured. Total proteins were obtained throughprotein lysis, the concentration of which was detected by BCA protein concentration Detection Kit (Thermo Fisher, 23227). Different proteins were isolated by SDS-PAGE and then transferred to PVDF membrane, which was followed by incubation with primary antibodies such as CD63, TSG101 and HSP70 and corresponding secondary antibodies. Photographs were obtained under a gel imaging development (TOCAN240, LCNJ2021076623).
4
Comprehensive Nanoparticle Characterization
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Transmission electron microscope H-7650 was purchased from Hitachi (Japan); Nanoparticle tracking analyzer was purchased from PMX (Germany); 0.22um lter was purchased from Millipore Corporation (USA); Size exclusion chromatography and ltration kit from Echo Biotech (China) Purchase; BCA protein concentration detection kit, cryostat centrifuge were purchased from Thermo Scienti c (USA); anti-CD9, CD63, Flotillin-1 and calnexin primary antibodies were purchased from CST (Cambridge, Massachusetts, USA); Hyaluronidase and polyethylene glycol (PEG6000) were purchased from Sigma (USA).
5
Comprehensive Nanoparticle Characterization
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Transmission electron microscope H-7650 was purchased from Hitachi (Japan); Nanoparticle tracking analyzer was purchased from PMX (Germany); 0.22um lter was purchased from Millipore Corporation (USA); Size exclusion chromatography and ltration kit from Echo Biotech (China) Purchase; BCA protein concentration detection kit, cryostat centrifuge were purchased from Thermo Scienti c (USA); anti-CD9, CD63, Flotillin-1 and calnexin primary antibodies were purchased from CST (Cambridge, Massachusetts, USA); Hyaluronidase and polyethylene glycol (PEG6000) were purchased from Sigma (USA).
6
Comprehensive Nanoparticle Characterization
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Transmission electron microscope H-7650 was purchased from Hitachi (Japan); Nanoparticle tracking analyzer was purchased from PMX (Germany); 0.22um lter was purchased from Millipore Corporation (USA); Size exclusion chromatography and ltration kit from Echo Biotech (China) Purchase; BCA protein concentration detection kit, cryostat centrifuge were purchased from Thermo Scienti c (USA); anti-CD9, CD63, Flotillin-1 and calnexin primary antibodies were purchased from CST (Cambridge, Massachusetts, USA); Hyaluronidase and polyethylene glycol (PEG6000) were purchased from Sigma (USA).
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