2 research outputs found
Development of a Simple Method for Protein Conjugation by Copper-Free Click Reaction and Its Application to Antibody-Free Western Blot Analysis
There are currently many methods available for labeling
proteins
in order to study their structure and function. However, the utility
of these methods is hampered by low efficiency, slow reaction rates,
nonbiocompatible
reaction conditions, large-sized labeling groups, and the requirement
of specific side chains such as cysteine or lysine. In this study,
a simple and efficient method for protein labeling was developed,
in which an azide-containing amino acid was introduced into a protein
and conjugated to a labeling reagent by strain-promoted azide–alkyne
cycloaddition (SPAAC). This method allowed us to label proteins
by simply mixing a protein and a labeling reagent in physiological
conditions with a labeling yield of approximately 80% in 120 min.
In addition, the specificity of SPAAC made it possible to analyze
the expression level of a protein quantitatively by simple mixing
and SDS-PAGE analysis with no need for antibodies or multistep incubations.
Because the genetic incorporation of the azide-containing
amino acid can be generally applied to any protein and the SPAAC reaction
is highly specific, this method should prove useful for labeling and
analyzing proteins