|Transfected Only||Mouse IgG1|
Immunoprecipitation of extracts from COS-7 cells transfected with a construct expressing a His-tagged protein (Tyro3-His), using Mouse IgG (Magnetic Bead Conjugate) #5873 (lane 2) or His-Tag (27E8) Mouse mAb (Magnetic Bead Conjugate) (lane 3). Lane 1 is 10% input. The western blot analysis was performed using His-Tag Antibody #2365.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: 408
Supplied in PBS Buffer (pH 7.2), 0.1% Tween® 20. Store at 4°C. Do not aliquot the antibodies.
His-Tag (27E8) Mouse mAb (Magnetic Bead Conjugate) detects 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.
All Species Expected
Monoclonal antibody is produced by immunizing animals with a 6xHis synthetic peptide.
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.
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