FLAG peptide

FLAG peptide is a multifunctional fusion tag for the purification of recombinant proteins. FLAG peptide maintances the natural folding of its fusing proteins. FLAG peptide can be removed by enterokinase, and eluted under non-denaturing conditions^[1]. In Vitro:Flag peptide is a highly acidic octapeptide that has a calcium-dependent epitope for monoclonal antibodies, allows elution under non-denaturing conditions, and can be fused to the N-terminus of the target protein. Flag peptide can be captured by the immunoaffinity column in the presence of Ca²⁺ and eluted by EDTA at low concentrations and neutral pH (thus close to physiological conditions)^[1][2].
Reprogramming factors in mammalian cells are purified by FLAG peptide^[3] (1) Transfected cells: HEK293 cells were transfected with FLAG-tagged reprogramming factors, and washed with PBS twice 24 h after transfection. (2) Collect cells: With NP40 cell lysis buffer (50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 5 mM EDTA, 1% NP-40, and protease inhibitors). Centrifuge the cell lysate at 14,500 rpm for 15 min at 4 °C to collect the soluble fraction. (3) Add Anti-Flag Affinity Gel(HY-K0217) to the supernatant and incubate in a cold room with rotation for 16 h. (4) Wash twice with PBS and add 0.1 mg/ml FLAG peptide to elute and retain the FLAG-tagged protein. (5) The eluted protein is dialysed with a dialysis tubing in dialysis buffer (PBS).