Octet® DYKDDDDK Biosensors
- High specificity to Flag®-tagged proteins for analysis of crude and purified samples
- High ligand loading capacity and sensitivity
- Wide dynamic range for quantification: 0.5 - 100 μg/mL
- Typical precision for quantitation: CV <10%
- Efficient and cost-effective regeneration for biosensor re-use up to 10 times
Overview
The Flag®-tag (also written FLAG®-tag or DYKDDDDK-tag) is a synthetic epitope/peptide tag not derived from natural proteins. It enables sensitive protein capture and detection across workflows like protein purification, ELISA, flow cytometry, immunofluorescence (IF), immunoprecipitation (IP), and Western blotting (WB). This octapeptide tag features the sequence NH2-DYKDDDDK-COOH (Asp–Tyr–Lys–Asp–Asp–Asp–Asp–Lys) with a built-in enterokinase cleavage site (DDDDK), allowing precise removal of the tag post-purification. Flag®-tagged proteins are specifically recognized by anti-Flag® antibodies, and the tag can be cloned in-frame at either the N- or C-terminus of a protein of interest in a DNA plasmid for recombinant expression via transfection.
Introduced by Hopp et al. in 1988 (1), the 1× Flag-tag comprises 8 amino acids with a molecular weight of ~1012.98 Da; a high-sensitivity 3× Flag variant is also widely used and commonly employs the optimized motif DYKDHDG–DYKDHDI–DYKDDDDK rather than a simple repeat. Whether for antibody-based detection, recombinant protein purification, or streamlined assay development, the Flag®-tag delivers robust, versatile performance from cloning to cleanup.
The Flag® tag is regarded as highly specific with a dissociation constant KD of about 100 nM for binding of the Flag® sequence to the corresponding antibody (2). Octet® DYKDDDDK Biosensors bind this short hydrophilic tag and can be used to study protein-protein interactions.
These biosensors offer high binding capacity, sensitivity, and a wide dynamic range. They can be regenerated up to 10 times for kinetic and quantitation assays while maintaining consistent and precise measurements. This makes them a cost-effective option for a wide range of high-throughput applications, including lead identification and optimization, cell line development, process development, and QC in both crude and purified protein samples.
(1) Hopp, T., Prickett, K., Price, V. et al. A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification. Nat Biotechnol 6, 1204–1210 (1988). https://doi.org/10.1038/nbt1088-1204
(2) Einhauer, A & Jungbauer, A. (2001). Affinity of the monoclonal antibody M1 directed against the FLAG peptide. Journal of chromatography. A. 921. 25-30. https://doi.org/10.1016/S0021-9673(01)00831-7
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Frequently Asked Questions
Use the FLAG®-tag when you need highly specific, antibody-based capture and clean detection of recombinant proteins, especially when purity matters more than yield.
Key reasons to use FLAG®-tag:
- High purity, low background: Anti-FLAG® antibody affinity is highly specific, typically yielding cleaner preparations than charge-based His-tag/IMAC methods (1). This is particularly valuable when contaminants co-purify with His-tags.
- Best for membrane proteins: Often recommended for membrane protein purification where achieving high purity is more critical than maximizing yield.
- Rapid, flexible purification: Works by fusing FLAG® to the N- or C-terminus and capturing with anti-FLAG monoclonal antibody on agarose or magnetic beads.
- Broad detection and assay support: The same anti-FLAG® antibodies can detect expression by utilizing Octet® BLI assays and are useful for immunoprecipitation workflows.
- Practical improvements in resins: Newer anti-FLAG® resins are more affordable, tolerate low pH elution, and have a longer usable lifespan, improving reusability and cost-effectiveness.
- Trade-off to note: FLAG®-tag generally gives lower yields than His-tag, but compensates with higher specificity and purity.
(1) Spriestersbach, Anne & Kubicek, Jan & Schaefer, Frank & Block, Helena & Maertens, Barbara. (2015). Purification of His-Tagged Proteins. Methods in enzymology. 559. 1-15. https://doi.org/10.1016/bs.mie.2014.11.003 .
Fuse the FLAG®-tag to either the N- or C-terminus of your protein (the optimal end is protein-dependent, so test both). For purification, capture the tagged protein with anti-FLAG® antibody-conjugated agarose or magnetic beads; using the FLAG®-tag in tandem can improve capture and purity, which is especially helpful for membrane proteins where purity outweighs yield. Follow the vendor’s protocol for binding, washing, and elution; elute competitively with FLAG peptide or via low pH if your resin supports it. Recent anti-FLAG® resins are more affordable, tolerate low-pH elution, and last longer.
The Octet® DYKDDDDK Biosensors are anti-FLAG®–coated and capture the FLAG-tagged protein as a clean, oriented ligand for interaction measurements. This antibody-based capture is highly specific, often preserves activity due to the small epitope, and can be regenerated with low pH. For good practice use controls: a non-FLAG® protein (negative control) and/or an isotype control surface to assess nonspecific binding.
FLAG is a registered trademark of Merck KGaA, Darmstadt, Germany and ANTI-FLAG is a trademark of Sigma-Aldrich Co. LLC.
