The protein composition of the OMVs was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, 2 mg of the protein derived was mixed with SDS-PAGE sample buffer (Invitrogen, USA), heated at 90 °C for 10 min after which 20 µL was placed on a 12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining and imaging. Standard protein markers (Beyotime Institute of Biotechnology, China) were used as a molecular weight marker, ranging from 10 to 150 kDa. Protein bands in the gel were analyzed using ImageJ software version 1.46 (NIH Research Services, Branch, Bethesda, Maryland, USA).
Standard protein markers
Manufactured by Beyotime
Sourced in China
Standard protein markers are a set of pre-stained proteins with known molecular weights used for estimating the size of unknown proteins in gel electrophoresis experiments. They serve as a reference to determine the approximate molecular weight of proteins separated by SDS-PAGE.
Automatically generated - may contain errors
3 protocols using standard protein markers
1
SDS-PAGE Analysis of OMV Proteins
2
SDS-PAGE Analysis of OMV Proteins
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Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the protein composition of the OMVs. The OMVs were mixed with SDS-PAGE sample buffer (Beyotime, Jiangsu, China) and immersed at 90 °C for 10 min. After that, the mixture was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was used for SDS-PAGE. Then, 20 µg samples were placed on a 12% (wt/vol) polyacrylamide gel. Finally, the gel was stained with Coomassie blue and imaged using a ChemiDoc system (Bio-Rad, Shanghai, China). Standard protein markers (Beyotime, Jiangsu, China) were used as molecular weight markers, ranging from 10 to 200 kDa.
3
SDS-PAGE Protein Separation Protocol
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SDS-PAGE was performed using a modified version of previously described methods (Alu'datt et al., 2012 ). Freeze-dried protein samples were formulated into the same concentration of protein solution and heated (95 °C, 5-10 min) in sample buffer (10% SDS, 0.5 M β-mercaptoethanol, 0.5 M Tris-HCl, pH 6.8, 2% glycerol, and 0.1% bromophenol blue). The acrylamide content for the stacking and resolving gels was 5 % and 12 %, respectively. A series of standard protein markers (16.0-270 kDa, Beyotime Biotechnology, Shanghai, China) was used in the electrophoresis to aid in the identification of protein bands in the samples. Electrophoresis was carried out on a vertical gel electrophoresis unit (Mini-Protean II; Bio-Rad Laboratories, Richmond, CA) at 15 mA, and bands were stained with Coomassie Brilliant Blue R-250. Destaining was performed using 40% methanol and 10% acetic acid. Densitometric analysis was performed on all bands on the SDS-PAGE gels using the Gel Imaging System (GelDoc XR+, Bio-Rad, USA).
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