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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.
Pack Size | Price | Availability | Quantity |
---|---|---|---|
1 mg | $66 | In Stock | |
5 mg | $153 | In Stock | |
10 mg | $226 | In Stock | |
25 mg | $380 | In Stock | |
50 mg | $565 | In Stock | |
100 mg | $793 | In Stock | |
200 mg | $1,130 | In Stock |
Description | 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. |
In vitro | 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] |
Alias | 3x FLAG peptide, 3x DYKDDDDK Peptide, 3X (DYKDDDDK) Peptide |
Molecular Weight | 3002.88 |
Formula | C123H176N30O58 |
Smiles | [H]N[C@@H](CC(O)=O)C(N[C@@H](CC1=CC=C(C=C1)O)C(N[C@H](C(N[C@@H](CC(O)=O)C(N[C@@H](CC(O)=O)C(N[C@@H](CC(O)=O)C(N[C@@H](CC(O)=O)C(N[C@H](C(N[C@@H](CC(O)=O)C(N[C@@H](CC2=CC=C(C=C2)O)C(N[C@H](C(N[C@@H](CC(O)=O)C(N[C@@H](CC(O)=O)C(N[C@@H](CC(O)=O)C(N[C@@H](CC(O)=O)C(N[C@H](C(N[C@@H](CC(O)=O)C(N[C@@H](CC3=CC=C(C=C3)O)C(N[C@H](C(N[C@@H](CC(O)=O)C(N[C@@H](CC(O)=O)C(N[C@@H](CC(O)=O)C(N[C@@H](CC(O)=O)C(N[C@H](C(O)=O)CCCCN)=O)=O)=O)=O)=O)CCCCN)=O)=O)=O)CCCCN)=O)=O)=O)=O)=O)CCCCN)=O)=O)=O)CCCCN)=O)=O)=O)=O)=O)CCCCN)=O)=O |
Relative Density. | no data available |
Sequence | Met-Asp-Tyr-Lys-Asp-His-Asp-Gly-Asp-Tyr-Lys-Asp-His-Asp-Ile-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys |
Sequence Short | MDYKDHDGDYKDHDIDYKDDDDK |
Storage | keep away from moisture | Powder: -20°C for 3 years | In solvent: -80°C for 1 year | Shipping with blue ice/Shipping at ambient temperature. |
Solubility Information | TBS (0.5M Tris-HCl, pH 7.4, with 1M NaCl): 20 mg/mL (6.66 mM), Sonication is recommended. |
Solution Preparation Table | |
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