Two-dimensional (2D) gel electrophoresis was carried out as previously described (Kurniyati et al., 2017 (link)). Equal amounts of purified PFs were resuspended in a rehydration buffer (5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10, 0.2% Bio-Lyte 3/10 Ampholyte, 40 mM Tris, and 0.0002% Bromophenol Blue) and then subjected to separation using 7 cm long pH 5→8 linear IPG strips. The first dimension of isoelectric focusing (IEF) was performed using PROTEAN IEF (Bio-Rad Laboratories, Hercules, CA), followed by equilibration according to the manufacturer's protocol. The second-dimension separation was carried out using sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis (SDS-PAGE) as described previously (Kurniyati et al., 2017 (link)). The resultant gels were subjected to Coomassie blue staining or immunoblotting analyses. The antibodies against T. pallidum FlaBs and T. denticola FlaA and DnaK are described in our previous publications (Kurniyati et al., 2017 (link), Kurniyati & Li, 2016 (link)). The stoichiometry of each flagellar filament protein was analyzed using Image Lab software from Bio-Rad (Bio-Rad).
Protocol detail
Proteomic Analysis of Flagellar Proteins
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2 thiourea
2d gel electrophoresis
Ampholyte
Antibodies
Bromophenol blue
Buffer
Chaps
Coomassie blue
Filament
Gels
Immunoblotting analyses
Pallidum
Protein
Rehydration
Sds page
Tris
Urea
Corresponding Organization : Virginia Commonwealth University
Other organizations : Yale University
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Variable analysis
independent variables
- Purified PFs resuspended in rehydration buffer
- First dimension of isoelectric focusing (IEF)
- Second-dimension separation using SDS-PAGE
- Separation of proteins using 2D gel electrophoresis
- Coomassie blue staining or immunoblotting analyses
- Stoichiometry of each flagellar filament protein
- Equal amounts of purified PFs
- Rehydration buffer composition (5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10, 0.2% Bio-Lyte 3/10 Ampholyte, 40 mM Tris, 0.0002% Bromophenol Blue)
- PH 5→8 linear IPG strips
- PROTEAN IEF system (Bio-Rad Laboratories)
- SDS-PAGE protocol as described previously (Kurniyati et al., 2017)
- Antibodies against T. pallidum FlaBs and T. denticola FlaA and DnaK (as described in previous publications)
- No negative controls were explicitly mentioned
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