Affinity purification resin binds the same epitope as Sigma's Anti-FLAG® M2 Antibody for DYKDDDDK-tagged fusion proteins. High yield and high purity. Calcium-independent protein preparation and Co-IP. Ready for batch/gravity, spin, and FPLC applications. ... Read more
PROTEINDEX™ Anti-DYKDDDDK Agarose 4 Fast Flow is an affinity chromatography medium for the purification of proteins that contain the peptide sequence DYKDDDDK (N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C). The ligand covalently immobilized on the matrix support is a recombinant mouse monoclonal IgG1 species that binds the same epitope as Sigma's Anti-FLAG® M2 Antibody, and it is highly specific for the peptide DYKDDDDK in a calcium-independent manner. Commonly known as the FLAG® epitope, the amino acid sequence DYKDDDDK has been used as an affinity tag to fuse at the N-terminal, C-terminal, or an internal location of a recombinant protein.
For protein purification, the ProteIndex™ Anti-DYKDDDDK Agarose 4FF resin is added to the sample. Following capture of the DYKDDDDK-tagged protein, the unbound and non-specifically bound proteins can be washed away. The bound target protein can then be eluted either with the competitive peptide under mild conditions or with the acidic glycine-HCl buffer.
Because the binding between the epitope and the antibody can occur under the native conditions, any proteins or other macromolecules that are able to interact with the tagged fusion protein will also be captured during the binding step. Therefore, the anti-DYKDDDDK 4FF affinity resin can be used in co-immunoprecipitation to analyze the protein complex that the target fusion protein is associated with.
The base matrix of ProteIndex™ Anti-DYKDDDDK Agarose 4FF is highly cross-linked 4% agarose, a robust support offering low background and high physical stability for chromatography procedures at various scales. The affinity resin is recommended for use in gravity/batch applications, spin purification columns, or cartridges for FPLC instruments.
- High selectivity for high purity (>90%)
- > 3 mg DYKDDDDK-tagged protein (50 kDa)/mL medium
- Stable coupling of ligand, low leakage; can be re-used to purify the same protein (3~5 repeated uses when acid elution is employed)
- Versatile - ready for gravity/batch, spin columns, and FPLC applications
Figure 1. Purification performance of ProteIndex™ Anti-DYKDDDDK 4FF affinity resins.
Whole cell lysate containing a DYKDDDDK-tagged fusion protein (~ 50 kDa) was loaded on the column packed with the anti-DYKDDDDK 4FF affinity resin. The column was washed with Binding/Wash Buffer (50 mM Tris，0.15 M NaCl，pH7.4). The target DYKDDDDK-tagged protein was partially eluted with 2 column volumes of 3x DYKDDDDK peptide (0.1 mg/mL, A) or the glycine-HCl buffer (B).
Shown in the SDS-PAGE gel images, from left to right fractions are: lysate (CL), flow-through (FT), eluate (E), and the supernatant of the boiled resin in the SDS-sample buffer (B).
This product can be packed into column for FPLC on automated systems. If you are interested in the pre-packed cartridge formats, talk to our customer support for further assistance.
|Ligand||Mouse monoclonal anti-DYKDDDDK IgG1, calcium-independent
|Binding capacity||≥ 3 mg DYKDDDDK-tagged protein (~ 50 kDa) per mL medium
|Matrix||4% highly cross-linked agarose supplied as 50% slurry|
|Particle size||45 μm - 165 μm with the average particle size being 90 μm
||0.3 MPa (3 bar)
|Recommended flow rate
||30 - 250 cm/hour|
||2 - 8°C|
||1x PBS with 0.02% NaN3
Product is shipped at ambient temperature. Upon receipt store at 2 - 8°C. Do not Freeze.
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