Hi colleagues,
I recently used the periodate oxidation method to conjugate HRP (horseradish peroxidase) to a recombinant protein I expressed and purified. The goal is to use this conjugate as a biorecognition element in a biosensor system (for ELISA-like detection or signal generation).
The HRP conjugation was done following the classical sodium periodate oxidation protocol, where oxidized HRP reacts with amine groups on the protein.
My question:
What are the best ways to validate that the conjugation was successful and that the conjugate is still functionally active (both enzymatically and as a biological receptor)?
Do you know the extinction coefficient for your recombinant protein Absorbance of 1mg/mL solution at 278nm wavelength?
When making HRP-antibody (IgG) conjugate you can work out the molar ratio of HRP to IgG using a spectrophotometer as follows: 1mg/mL of IgG give an optical density of 1.4 at 278nm wavelength. For HRP, 1mg/mL has an optical density of 0.75 at 278nm and an optical density of 2.2 at 403nm. When you have made your conjugate and assuming you have removed unbound enzyme and IgG, measuring the optical density of the conjugate at both wavelengths will allow you to workout the molar ratio HRP:IgG.
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