title: Development of a simple method for protein conjugation by copper-free click reaction and its application to antibody-free Western blot analysis.
abstract: 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.
authors.affiliation: Department of Chemistry, Sogang University, Seoul 121-742, Republic of Korea.
authors.forename: Sohye
authors.initials: S
authors.lastname: Jang
authors.forename: Kalme
authors.initials: K
authors.lastname: Sachin
authors.forename: Hui-jeong
authors.initials: HJ
authors.lastname: Lee
authors.forename: Dong Wook
authors.initials: DW
authors.lastname: Kim
authors.forename: Hyun Soo
authors.initials: HS
authors.lastname: Lee
mesh_terms: D000480:Alkynes / Q000737:chemistry*; D000596:Amino Acids / Q000737:chemistry*; D001386:Azides / Q000737:chemistry*; D015153:Blotting, Western / Q000379:methods*; D057930:Click Chemistry; D003300:Copper; D003500:Cyclization; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure
chemical_list: D000480:Alkynes; D000596:Amino Acids; D001386:Azides; D003300:Copper; D005982:Glutathione Transferase