|Transfected Only||Rabbit IgG|
Immunoprecipitation of extracts from COS-7 cells, mock transfected (lanes 1, 3, and 5; -) or transfected with a construct expressing CBP/DYKDDDDK-tagged full-length human Arrb1 (lanes 2, 4, and 6; +) using Rabbit (DA1E) mAb IgG XP® Isotype Control (Sepharose® Bead Conjugate) #3423 (lanes 1 and 2) or DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) (Sepharose® Bead Conjugate) (lanes 3 and 4). Lanes 5 and 6 are 10% input. Western blot analysis was performed using DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) (HRP Conjugate) #86861.Learn more about how we get our images.
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
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.
DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma's Anti-Flag® M2 Antibody) (Sepharose® Bead 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.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. Anti-FLAG is a registered trademark of Sigma-Aldrich Biotechnology. Sepharose is a registered trademark of GE Healthcare.
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|70569S||400 µl||$ 299.0|