Flow cytometric analysis of COS-7 cells, untransfected (blue) or transfected with a DYKDDDDK-tagged fusion protein (green), using DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) (Pacific Blue™ Conjugate).
This Cell Signaling Technology antibody is conjugated to Pacific Blue™ fluorescent dye and tested in-house for direct flow cytometry in monkey cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) #14793.
Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2020
Protocol Id: 407
DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) (Pacific Blue™ Conjugate) detects exogenously expressed DYKDDDDK proteins in cells. The antibody recognizes the DYKDDDDK peptide, which is the same epitope recognized by Sigma's Anti-FLAG® antibodies, fused to either the amino-terminus or carboxy-terminus of the target protein.
All Species Expected
Monoclonal antibody is produced by immunizing animals with a synthetic DYKDDDDK peptide.
Epitope tags are useful for the labeling and detection of proteins using immunoblotting, immunoprecipitation, and immunostaining techniques. Because of their small size, they are unlikely to affect the tagged protein’s biochemical properties.
The DYKDDDDK peptide has been used extensively as a general epitope tag in expression vectors. This peptide can be expressed and detected with the protein of interest as an amino-terminal or carboxy-terminal fusion (1).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Anti-FLAG is a registered trademark of Sigma-Aldrich Biotechnology.
Pacific Blue is a trademark of Molecular Probes, Inc.