|5000 bead||Endogenous||Rabbit IgG|
Immunoprecipitation of extracts from HeLa cells, untreated or TPA-treated, using Rabbit (DA1E) mAb IgG XP® Isotype Control (Sepharose® Bead Conjugate) (Lanes 1 and 2) or Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb (Sepharose® Bead Conjugate) #3510 (Lanes 3 and 4). The blot was probed using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) Mouse mAb #9106.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
Directions for Use: Add 10 μl of well-vortexed beads to 200 μl of cell lysate at 1 mg/ml in 1X Cell Lysis Buffer (10X) #9803. See protocol for more details.
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.
Rabbit (DA1E) mAb IgG XP® Isotype Control is not directed against any known antigen. It functions as a control for rabbit IgG monoclonal antibodies.
Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (whole molecule) from Cell Signaling Technology is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated Sepharose® beads. Rabbit (DA1E) mAb IgG XP® Isotype Control (Sepharose® Bead Conjugate) is useful to determine non-specific immunoprecipitation complexes.
Control antibodies are used to estimate the non-specific binding of target primary antibodies due to Fc receptor binding or other protein-protein interactions.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc.
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|3423S||400 µl (40 immunoprecipitations)||$124.00.0|