To biotinylate the protein backbone of mucin, 100 μL of 100 mM N-biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester (BNHS) (Tokyo Chemical Industry) in dimethylsulfoxide and 150 μL of PBS were added to 250 μL of 5% (w/v) PGM stock solution. After incubation for 4 hr at room temperature, unreacted biotin was separated from the biotinylated mucin using a Zeba spin desalting column equilibrated with 50 mM sodium bicarbonate (pH 9.6). The eluate was collected to give a final concentration of 25 mg/mL and stored at 4°C until use.
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Biotinylation of Mucin Backbone
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Corresponding Organization : Kurume University
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Variable analysis
independent variables
- Concentration of N-biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester (BNHS) (100 mM)
- Biotinylation of the protein backbone of mucin
- Volume of BNHS solution (100 μL)
- Volume of PBS (150 μL)
- Volume of 5% (w/v) PGM stock solution (250 μL)
- Incubation time (4 hours)
- Incubation temperature (room temperature)
- Purification method (Zeba spin desalting column equilibrated with 50 mM sodium bicarbonate, pH 9.6)
- Final concentration of biotinylated mucin (25 mg/mL)
- Storage conditions (4°C)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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