Protein was extracted from human stellate ganglia that were processed individually. The stellate samples were homogenized in ice-cold dPBS without Ca2+ or Mg2+ and the protein within the samples was normalized using a standard protein assay (BioRad DC). Enzyme immuno assays (EIA) or enzyme-linked immunosorbent assays (ELISA) were conducted to detect the presence of the following proteins of interest in human stellate ganglia: AGT (CSB-E08564h, Cusabio), renin (dren00, R&DSystems), AngII (RAB0010-1KT, Sigma), ACE2 (LS-F5886, LSBio), Ang1-7 (CSB-E14242h). Briefly, standards or samples (2–3 repeats) were incubated in 96-well plates and each assay was carried out as per the manufacturer's instructions. The absorbance or fluorescence from each well was measured within 5 min at the appropriate wavelength and the background was subtracted from primary absorbance values (Infinite F500, Tecan). The expression of each relevant protein was quantified using a standard curve generated from the supplied standards (GraphPad Prism, v7).
Standard protein assay
Manufactured by Bio-Rad
The Standard Protein Assay is a colorimetric-based method for quantifying the total protein concentration in a sample. It measures the absorbance of the sample at a specific wavelength, which is proportional to the amount of protein present. This assay provides a reliable and consistent way to determine the protein content in various biological samples.
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Lab products found in correlation
Anti β actin monoclonal antibody, by Merck Group (2 mentions)
Horseradish peroxidase conjugated goat anti mouse immunoglobulin g, by Bio-Rad (2 mentions)
Prism v7, by GraphPad (1 mentions)
Infinite f500, by Tecan (1 mentions)
Qproteome mammalian protein prep kit, by Qiagen (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Cocktail of proteinase inhibitors, by Roche (1 mentions)
9 protocols using standard protein assay
1
Quantifying Stellate Ganglia Protein Markers
2
Exosome Isolation from Serum
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Isolation of exosomes from serum samples were performed as previously described method [16 (link)]. Briefly, thawed serum samples were centrifuged at 10,000 × g for 30 min at 4 °C to completely eliminate cellular debris. Thereafter, the supernatant was centrifuged at 100,000 × g for 70 min at 4 °C. The pellets were washed with phosphate-buffered saline (PBS), ultracentrifuged, and re-suspended in PBS. Subsequently, isolated exosomes were quantified using a standard protein assay (Bio-Rad Laboratories, Hercules, CA) and stored at − 80 °C until needed.
3
Protein Analysis of Injected Mouse Liver
4
Western Blot Analysis of HCV Proteins
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The HCV infected or transfected Huh7.5 cells were lysed in a radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate) containing a cocktail of protease inhibitors (Roche). The total protein for each sample was measured using a standard protein assay (Bio-Rad). Twenty micrograms of total protein of each sample was analyzed by 8% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk. HCV proteins were detected with a monoclonal antibody specific for NS3, horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (Bio-Rad) and a chemiluminescence substrate (Pierce). Actin was used as a control and was detected with an anti-β-actin monoclonal antibody (Sigma).
5
Nitrogenase Activity in Bacterial Suspensions
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Nitrogenase activity was determined with bacterial suspensions incubated to an OD600 of 0.1 in N-free minimal medium (0.5% oxygen and 10% acetylene) at 30°C (26 (link)). Protein concentrations were determined using a standard protein assay (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard. The specific activity of nitrogenase was expressed as nmol ethylene per hour per milligram of protein. Each experiment was repeated at least three times.
6
Nitrogenase Activity Quantification Assay
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Nitrogenase activity was determined according to the previously described derepression protocol (Desnoues et al. 2003 (link)). Bacterial suspensions were incubated at an OD600 of 0.1 in N-free minimal lactate medium (0.5% oxygen and 10% acetylene) at 30 °C. Protein concentrations were determined using a standard protein assay (Bio–Rad, Hercules, CA) with bovine serum albumin as a standard. The specific activity of nitrogenase was expressed as nmol ethylene per hour per milligram of protein. Each experiment was repeated at least three times.
7
Protein Concentration Determination
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Sample protein concentrations were determined using a standard protein assay (Bio-Rad, Hercules, CA) based on the Bradford assay method. The linear regression method was used to calculate the protein concentration of the individual samples, which were normalized before loading.
8
Immunoblotting Analysis of HCV Proteins
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The HCV-transfected Huh 7.5 cells were lysed in a radioimmuno-precipitation assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1% Nonidet P40, 0.5% sodium deoxycholate) containing a cocktail of proteinase inhibitors (Roche). The total protein for each sample was measured with a standard protein assay (Bio-Rad). Twenty-five micrograms of total protein for each sample was analyzed by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked by incubating them with 5% skim milk. HCV proteins were detected with monoclonal antibodies specific to NS5A, horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (Bio-Rad) and a chemiluminescence substrate (Pierce). β-Actin was used as a control and was detected with an anti-β-actin monoclonal antibody (Sigma).
9
Nitrogenase Activity Determination Protocol
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Nitrogenase activity was determined according to the derepression protocol previously described (Desnoues et al. 2003 (link)). Bacterial suspensions were incubated at an OD6 00 of 0.1 in N-free minimal lactate medium (0.5% oxygen and 10% acetylene) at 30°C. Protein concentrations were determined using a standard protein assay (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard. The speci c activity of nitrogenase was expressed as nmol ethylene per hour per milligram of protein. Each experiment was repeated at least three times.
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