Recently, I conducted an experiment to compare the use of EDC versus passive adsorption.
I attempted to immobilize goat anti-rabbit HRP secondary antibody to Carboxylated polystyrene beads via EDC and NHS linking protocol.
As the beads were polystyrene, we used centrifugation to clean the beads in between each step. Otherwise, we followed ThermoFisher's DynaBeads protocol. After immobilizing the ligands we added 100ul of HRP substrate to each sample and measured the luminance using Molecular Device's ID3.
The Materials used are as follows:
- 3um Carboxylated Beads (dyed red) from Polyscience
- ThermoFisher's Pierce Premium EDC
- 25mM MES Buffer (6.33 pH)
- 50mg/ml EDC
- 50mg/ml NHS
Antibody and Detection
- Goat Anti-Rabbit HRP Secondary Antibody
- HRP substrate
However, when measuring the luminance of the HRP substrate the passive adsorption seemed to consistently provide significantly brighter results, suggesting that the EDC linker is not as effective as I thought.
Further, when testing different concentrations of EDC I noticed that luminance decreased as the concentration of EDC increased.
These results seem consistently odd and I'm not sure what to make of them. Looking for advice, as I am completely new to this procedure.