Protein A coated surfaces

General procedure for binding Immunoglobulins to Protein A coated surface

  1. dilute immunoglobulin (sample) to 0,1- 5 µg/ml in an appropriate neutral pH buffer or slightly basic (8 - 8.5)
  2. proceed with incubation: conditions depend on biomolecule structure and purity
  3. wash four times to remove the unbound material
  4. proceed with your specific test:
    • to point out the bound immunoglobulin
    • to use the bound immunoglobulin to point out or to bind the specific antigen

example of test: binding capacity towards biotinylated HIgG

  1. Add 100 µl of different concentrations of biotinylated HIgG (from 1 to 0.015 µg/ml) to the wells of Protein A coated plate and incubate 30 minutes at room temperature. Add the same solutions to Albumin coated plate as comparison for evaluate the specificity of binding. Leave blank wells as control
  2. Empty the wells and wash with 0,1M PBS pH 7,2 + 0,05% Tween® 20 four times
  3. Add 100 µl /well of Streptavidin-Pod ( 160 ng/ml) and incubate for 30 minutes at room temperature
  4. Empty the wells and wash with 0,1M PBS pH 7,2 + 0,05% Tween® 20 four times
  5. Add 100 µl /well of TMB substrate solution and incubate for 10 minutes at room temperature
  6. Stop the substrate reaction by adding 100 µl of sulphuric acid 1 N and read the optical density values at 450 nm.