Immunoprecipitation of COS-7 cells transfected with a construct expressing HA-tagged Akt using HA-Tag (C29F4) Rabbit mAb (Magnetic Bead Conjugate) (Lane 2) or Rabbit (DA1E) mAb IgG XP® Isotype Control (Magnetic Bead Conjugate) #8726 (Lane 3). Lane 1 is 10% lysate. The western blot was probed using HA-Tag (6E2) Mouse mAb (HRP Conjugate) #2999.
This Cell Signaling Technology antibody is immobilized by the covalent reaction of formylbenzamide-modified antibody with hydrazide-activated magnetic bead. HA-Tag (C29F4) Rabbit mAb (Magnetic Bead Conjugate) is useful for immunoprecipitation assays of HA-tagged recombinant proteins.
Supplied in PBS Buffer (pH 7.2), 0.1% Tween® 20. Store at 4°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: 408
HA-Tag (C29F4) Rabbit mAb (Magnetic Bead 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.
XP is a registered trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.