Protein G coated - Binding capacity test

  1. Add 100 μl of different concentrations of human IgG (from 0.5 to 10 μg/ml) mixed with a constant amount of human IgG biotinylated (0.01 μg/ml) to the wells of Protein G coated plate and incubate for 30 minutes at room temperature.
  2. Empty the wells and wash with 0.1 M PBS pH 7.2,0.05% Tween® 20 four times
  3. Add 100 μl /well of Streptavidin-HRP (BioSpa product code SB01-61, diluted 1:20.000) and incubate for 30 minutes at room temperature.
  4. Empty the wells and wash with 0.1 M PBS pH 7.2,0.05% Tween® 20 four times
  5. Add 100 μl /well of TMB substrate solution and incubate 15 minutes at room temperature.
  6. Stop the substrate reaction by adding 100 μl /well of sulphuric acid 1 N and read the optical density values at 450 nm

The data show that a plateau has got starting with an IgG concentration of 8.0μg/ml.

This concentration means the well binding capacity we can express as:

Figure 1.