DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. Often it is also used in a 3X FLAG format (3x DYKDDDDK Tag TFA) for purifying difficult proteins that accumulate in low abundance [1][2].
HEK293 cells transiently transfected with HARS-expressing plasmids were harvested and lysed in CelLytic M buffer containing mammalian protease inhibitor cocktail for 20 min at 4 °C. FLAG-tagged HARS is purified by binding to an anti-DYDDDDK resin per manufacturer protocol and eluted by competition with 3X-DYKDDDDK peptide in buffer containing 50 mM Tris-HCl pH 7.4, and 150 mM NaCl (purification of human HARS)[2].
[1]. Mishra V. Affinity Tags for Protein Purification. Curr Protein Pept Sci. 2020;21(8):821-830.
[2]. Abbott JA, et al. The Usher Syndrome Type IIIB Histidyl-tRNA Synthetase Mutation Confers Temperature Sensitivity. Biochemistry. 2017;56(28):3619-3631.