Protein thiol is a major redox signaling in the cellular processes. Modification of protein thiol by oxidative stress associates with the redox state in the cells. The approach of this kit is detecting the number of thiol residues accurately in order to study the redox state in oxidative stress environment.
Fig. 1 Redox States of Thiol group(s) in Protein
Mechanism of Thiol Modification with Protein-SHifter:
Fig. 2 Maleimide group of the Protein-SHifter binds to a free thiol group of a protein. The conjugate consists of a unique molecular weight that clearly visualized in electrophoresis.
Visualization of Redox State of Thiol Residues in a Protein:
Fig. 3 One molecule of Protein-SHifter binds to one molecule of free-thiol in protein. The number of the conjugation is directly proportional to the number of free thiol as clear band-shift.
Linearity of mobility shifts derived from Protein SHifter comparing with PEG-Maleimide (PEG-Mal)
*Conjugation with Thioredoxin
Clear band-shift observed from Protein SHIfter comparing with PEG-Mal.
Detection of Redox State of Thiols residues in GAPDH
1) S. Hara, T. Nojima, K. Seio, M. Yoshida and T. Hisabori, “DNA-maleimide: An improved maleimide compound for electrophoresis-based titration of reactive thiols in a specific protein” Biochim. Biophys. Acta, 2013, 1830(4), 3077.
2) S. Hara, Y. Tatenaka, Y. Ohuchi and T. Hisabori, “Direct determination of the redox status of cysteine residues in proteins in vivo”, Biochem. Biophys. Res. Commun., 2015, 456, (1), 339.