3× FLAG peptide is a FLAG peptide tag composed of three repeated Asp-Tyr-Lys-Xaa-Xaa-Asp motifs, used for protein identification and purification.

3× FLAG peptide is often used for elution of anti-Flag resins, magnetic beads, etc. Take the steps of purifying human histidine-tRNA synthetase (HARS) as an example::
1. HEK293 cells transiently transfected with HARS expression plasmid were harvested and lysed in CelLytic M buffer containing mammalian protease inhibitor cocktail at 4 °C for 20 min.
2. FLAG-tagged HARS was purified by binding to anti-DYDDDDK resin and eluted by competition with 3× FLAG peptide in a buffer containing 50 mM Tris-HCl (pH 7.4) and 150 mM NaCl.
3. The isolated FLAG-HARS was further purified using HiTrapQ HP column (GE Healthcare) and eluted with NaCl gradient to 150–500 mM.
4. Fractions containing HARS were identified by SDS-PAGE, pooled and dialyzed at 4°C into a standard buffer consisting of 50 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl2 and 5 mM β-mercaptoethanol (β-ME).
5. After dialysis, samples were concentrated using Amicon Ultra-4 centrifugal filters (Millipore) and then diluted by adding 80% glycerol to a final glycerol concentration of 40%.
6. Protein concentration was determined by A280 and stored at −20°C. The HARS content of the preparations was greater than 99% as analyzed by SDS-PAGE (data not shown).
7. The activity of each enzyme preparation was determined by active site titration26,27, monitoring the appearance of α-labeled 32P-AMP in the presence of histidine under pre-steady-state conditions with rapid chemical quenching.
Prepare two syringes: in one syringe, incubate the enzyme at a concentration of 5 μM with a standard buffer containing saturated histidine, 5 mM MgCl2, and 8 U/mL pyrophosphatase (PPiase); in the other syringe, incubate ATP with a standard buffer.
8. Quench the reaction with 400 mM NaOAc (pH 4.5) and 0.1% SDS, and analyze the products by thin layer chromatography.[1]