One of the key elements of bioconjugation is knowing the characteristics of the biopolymers you are dealing with and quantifying their functional groups. For example, if a Cys is involved in a bioconjugation reaction, it is good practice to determine how much reactive Cys is in the biopolymers. Free thiols are easily oxidized in air. In another example, if a drug is conjugated to the protein through a surface amine, by quantifying the free amine groups prior to the conjugation reaction and after the conjugation reaction, you can determine the drug loading.

Typical examples of bioassays we do:

TNBS assay for amine: Standard colorimetric assay for NH2 quantification according to Snyder and Sobocinsky.

Ellman’s assay for thiol: Standard colorimetric assay for SH quantification based on Ellman’s assay.

Biotinylation determination: Estimation of protein biotinylation level using the HABA method.

BCA protein assay: For protein quantification.

Indirect Ellman’s assay: Standard colorimetric assay to evaluate thiol-reactive biopolymers based on indirect Ellman’s assay.

TNBS assay for acylating groups: Standard colorimetric assay to evaluate the activation of acylating groups (NHS etc.).