Western Blot
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The Recombinant immunogen was migrated by 12% SDS-PAGE and transferred to a 0.2 um PVDF membrane.
- After blocking the membrane in blocking buffer (5% milk powder in 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 0.1% (v/v) Tween 20 , the membrane was incubated with biotinylated primary antibody (0.1ug/ml) at 4°C overnight.
- Enhanced chemiluminescence (ECL) reagents were used to visualize
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Sandwich ELISA
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96-well maxisorp plates were coated with the capture antibody overnight, then blocked with 5% FCS in PBS for 1 hour, then washed.
- Then, the protein standard was added, and plates were incubated for 2 hrs at 37°C.
- Plates were washed and incubated with a biotinylated detection antibody for 1 hour at 37°C.
- After washing, plates were incubated with HRP-conjugated streptavidin for 30 min at room temperature and washed again.
- Plates were developed using the tetramethylbenzidine peroxidase substrate system and absorbance was measured at 450-615 nm using an automated plate reader.
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