Immunoprecipitation of phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) from NIH/ 3T3 cell extracts, treated with Human Platelet-Derived Growth Factor AA (hPDGF-AA) #8913 (10 min), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb (Biotinylated) #4094 and Streptavidin (Magnetic Bead Conjugate) (lane 3). Lysate and Streptavidin (Magnetic Bead Conjugate) are shown in lane 2, demonstrating streptavidin bead binding. Lane 1 shows 10% lysate. Western blot analysis was performed using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) Mouse mAb #9106.
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.
For bead washing and subsequent elution of immunocomplexes, the beads can be separated from solution using our 6-Tube Magnetic Separation Rack #7017. Place the tubes containing the beads in the Magnetic Separation Rack and wait 1 to 2 minutes for the solution to clear before carefully removing the supernatant. Remove the tubes from the Magnetic Separation Rack, add new solution and resuspend the beads by gently vortexing or rocking the tube.
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
Streptavidin (Magnetic Bead Conjugate) is useful for the precipitation of biotinylated proteins (1,2). Recombinant streptavidin is immobilized by the covalent binding of primary amino groups with formylbenzamide-modified magnetic bead.
Streptavidin is a 53,000 dalton tetrameric protein purified from the bacterium Streptomyces avidinii (3). Each subunit binds to biotin with extremely high affinity. Because of its strong non-covalent interaction with biotin, streptavidin can be used to isolate biotinylated proteins (1,2).
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