The original DYKDDDDK peptide (the same epitope recognized by Sigma's Anti-FLAG®** antibodies) was an 11-amino-acid leader peptide of the gene-10 product from bacteriophage T7 fused at the amino-terminus of GAL4 (yeast transcription factor). The binding reaction of this tag to the anti-DYKDDDDK antibody is calcium dependent, so proteins can frequently be eluted from an affinity matrix by buffers containing EDTA. It also allows for the tag to be placed at the amino-terminus, at the carboxy-terminus, or in association with other tags.
Order DYKDDDDK peptide ($50 per mg) from here.
LifeTein's anti-DYKDDDDK monoclonal antibody was developed based on the most widely used hydrophilic octapeptide: DYKDDDDK. Order DYKDDDDK peptide ($50 per mg) from here. LifeTein's anti-DYKDDDDK renders higher affinity and higher binding capacity than Sigma's antibody. Please see the following table for technical details.
Comparison of anti-DYKDDDDK affinity gel with commercially available M2 affinity gel
| LT0420 |
| 411005100203 |
| Mouse IgG2b |
| FG4R |
| 50 μg, 1μg/μl |
| DYKDDDDK (FLAG*) synthetic peptide conjugated to KLH. Order DYKDDDDK peptide ($50 per mg) from here. |
| Recognizes the N-terminal, C-terminal or internal DYKDDDDK-tagged fusion proteins |
| Protein A affinity chromatography from mouse ascites fluid |
| Dot, ELISA, IS, IP, WB |
| WB (with ECL): 1-5ng of DYKDDDDK-tagged fusion proteins |
| WB (with ECL): 1:1000-3000 dilution (incubate for 1 hour at room temperature)
IS: 1:500-2000 dilution
For best results with other assays (e.g.: Dot, ELISA, IP, etc), please determine optimal working dilution by titration test |
| Store at -20°C for up to 2 years. Centrifuge after first thaw to maximize product recovery. Aliquot to avoid repeated freeze-thaw cycles. Store aliquots at 4°C for 1-2 weeks |
| 
Fig. 1:1000 (1µg/mL) Ab dilution probed against HEK293 cells transfected with DYKDDDDK-tagged protein vector; untransfected (1) and transfected (2) |
Order DYKDDDDK peptide ($50 per mg) from here.
*FLAG is a registered trademark of Sigma-Aldrich Co. LLC
**ANTI-FLAG is a registered trademark of Sigma-Aldrich Co. LLC
