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| Targets |
The tag's target is the protein of interest it is fused to, as well as anti-FLAG antibodies. The DYKDDDDK peptide (FLAG) is an epitope tag that binds specifically to high-affinity anti-FLAG antibodies. The 3x FLAG format has higher affinity and specificity than a single FLAG tag, allowing for robust detection and purification of fusion proteins, especially those that are low in abundance.
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| ln Vitro |
After being temporarily transfected with plasmids expressing HARS, HEK293 cells were collected and lysed for 20 minutes at 4 °C in CelLytic M solution containing a mammalian protease inhibitor cocktail. According to manufacturer instructions, FLAG-tagged HARS is purified by binding to an anti-DYDDDDK resin and eluting by competition with 3X-DYKDDDDK peptide in a buffer containing 50 mM Tris-HCl pH 7.4 and 150 mM NaCl (purification of human HARS)[2].
The in vitro activity is its ability to bind to anti-FLAG antibodies with high specificity and affinity. The 3x FLAG tag is often used to purify difficult proteins that accumulate in low abundance. In Western blotting or ELISA, the tag allows for the detection of the fused protein even when the antibody for the protein is not available. |
| ln Vivo |
In vivo applications are limited; however, it can be used to generate stable cell lines expressing 3x FLAG-tagged proteins for studying protein function, localization, and protein-protein interactions within living cells. The tag itself has no therapeutic in vivo activity but is a valuable tool for studying protein biology in model organisms.
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| Enzyme Assay |
No enzyme binding assays are performed. The interaction is typically measured using surface plasmon resonance (SPR) or ELISA to quantify binding affinity (Kd) between the FLAG tag and anti-FLAG antibodies. Alternatively, pulldown assays using anti-FLAG antibody-conjugated beads are used to measure binding capacity from complex lysates.
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| Cell Assay |
Cell-based assays involve transfecting cells with a plasmid encoding the gene of interest fused to the 3x FLAG tag. After 24-72 hours, cells are lysed, and the FLAG-tagged protein is immunoprecipitated using anti-FLAG antibody beads. The bound protein is eluted and analyzed by SDS-PAGE and Western blotting to confirm expression and protein-protein interactions.
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| Animal Protocol |
The 3x FLAG Tag is used in animal models to study protein function in vivo. Transgenic animals or xenograft models expressing the 3x FLAG-tagged protein are generated. Tissues are harvested, and the tagged protein is immunoprecipitated or detected by immunofluorescence (IF) using anti-FLAG antibodies to analyze tissue-specific expression and localization of the protein of interest.
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| ADME/Pharmacokinetics |
The 3x DYKDDDDK Tag TFA has a molecular formula C125H177F3N30O60 and molecular weight of 3116.90. It is stable as a powder at -20degC for 3 years and is soluble in water (H2O) at ≥ 100 mg/mL (32.08 mM). Storage in solution at -80degC for up to 6 months is recommended for long-term stability.
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| Toxicity/Toxicokinetics |
The 3x FLAG peptide is generally considered non-toxic at the concentrations used for research. It is a small peptide tag that does not exhibit cytotoxic or immunogenic effects when expressed in cells. The TFA counterion is non-toxic at standard concentrations. The tag is a standard research reagent and not intended for human use.
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| References |
[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. |
| Additional Infomation |
3x DYKDDDDK Tag TFA is a research-grade chemical tool used in molecular biology. It is the standard 3X FLAG format for purifying difficult proteins that accumulate in low abundance. This tag is for research use only and is not a drug, diagnostic, or therapeutic. It has no FDA approval for human use and is strictly a reagent for protein science.
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| Molecular Formula |
C125H177F3N30O60
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| Molecular Weight |
3116.90
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| Related CAS # |
3x DYKDDDDK Tag;921765-76-6
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| Appearance |
White to off-white solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
H2O :≥ 100 mg/mL (~32.08 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.3208 mL | 1.6042 mL | 3.2083 mL | |
| 5 mM | 0.0642 mL | 0.3208 mL | 0.6417 mL | |
| 10 mM | 0.0321 mL | 0.1604 mL | 0.3208 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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