Flow cytometric analysis of 293T cells, mock transfected (blue) or transfected with a construct expressing an HA-tagged protein (green), using HA-Tag (C29F4) Rabbit mAb (PE-Cy7® Conjugate) (solid lines) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE-Cy7® Conjugate) #97492 (dashed lines).
This Cell Signaling Technology antibody is conjugated to phycoerythrin in combination with cyanine 7 (PE-Cy7®) and tested in-house for direct flow cytometric analysis in human cells.
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
HA-Tag (C29F4) Rabbit mAb (PE-Cy7® Conjugate) detects exogenously expressed proteins containing the HA epitope tag.
All Species Expected
Monoclonal antibody is produced by immunizing animals with a synthetic peptide containing the influenza hemagglutinin epitope (YPYDVPDYA).
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 HA tag is derived from an epitope of the influenza hemagglutinin protein which has been used extensively as a general epitope tag in expression vectors (1).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Cy and CyDye are registered trademarks of GE Healthcare.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.