Immunoprecipitation of His-tagged Akt1 (Akt1-His) from COS cell extracts. Lane 1 is His-Tag (D3I10) XP® Rabbit mAb (Sepharose® Bead Conjugate) and lane 2 is 10% input. Western blot analysis was performed using His-Tag (D3I10) XP® Rabbit mAb (HRP Conjugate) #12688.
This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated Sepharose® beads.
His-Tag (D3I1O) XP® Rabbit mAb (Sepharose® Bead Conjugate) is useful for the immunoprecipitation of His-tagged proteins and peptides. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated His-Tag (D3I1O) XP® Rabbit mAb #12698.
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 antibody.
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
His-Tag (D3I1O) XP® Rabbit mAb (Sepharose® Bead Conjugate) recognizes recombinant proteins containing the 6xHis epitope tag. The antibody recognizes the 6xHis 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 corresponding to residues of the 6xHis epitope tag.
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.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Sepharose is a registered trademark of GE Healthcare.