A method for parallel microscale protein labeling and precise control over the average degree of labeling (aDoL).

ABSTRACT: A widely used approach for protein conjugation is through the lysine residues reacting with NHS- or other active esters. However, it is a challenge to precisely control the degree of labeling (DoL) due to the instability of active ester and variability of reaction efficiencies. Here, we provide a protocol for better control of aDoL using existing Copper-free Click Chemistry reagents. It is a two-step reaction with one purification in between. Briefly, proteins of interest were first activated with azide-NHS. After removing unreacted azide-NHS, the protein-N₃ is then reacted with a limited amount of complementary click tag. Our studies have shown the click tag will fully react with the protein-N₃ after 24 h' incubation, and therefore does not require additional purific

SUBMITTER: Ruan Q 

PROVIDER: S-EPMC10238424 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature