Immunoprecipitation of COS cell extracts, untransfected or transfected with HA-Akt protein, using HA-Tag (C29F4) Rabbit mAb (Sepharose® Bead Conjugate) (lanes 1 and 2) and Rabbit (DA1E) mAb IgG XP® Isotype Control (Sepharose® Bead Conjugate) #3423 (lanes 3 and 4). The western blot was probed using HA-Tag (C29F4) Rabbit mAb #3724.
This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated Sepharose® beads. HA-Tag (C29F4) Rabbit mAb (Sepharose® Bead Conjugate) is useful for the immunoprecipitation of HA-tagged recombinant proteins.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol. Store at –20°C. Do not aliquot the antibodies.
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
10X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin
NOTE: CST recommends adding 1 mM PMSF (#8553) before use*.
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2007
Protocol Id: 27
HA-Tag (C29F4) Rabbit mAb detects recombinant proteins containing the HA epitope tag. The antibody recognizes the HA-tag fused to either the amino or carboxy terminus of targeted proteins in transfected cells.Species Reactivity:
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. Due to 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.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.