Immunoprecipitation of COX IV from HeLa cells using COX IV (4D11-B3-E8) Mouse mAb #11967 and Protein G Magnetic Beads. Western blot analysis was performed on the 10% input control (lane 1), IP pellet (lane 2) and Mouse (G3A1) mAb IgG1 Isotype Control #5415 (at matched concentration) (lane 3) using COX IV (4D11-B3-E8) Mouse mAb #11967.
Supplied in PBS, 0.1% BSA, 0.004% EDTA and 0.075% sodium azide. Store at 4°C. This product is stable for 12 months. Do not freeze, dry or centrifuge beads. This may cause irreversible aggregation and decreased binding capacity.
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein G magnetic separation.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein G Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.
IMPORTANT: Pre-wash #70024 magnetic beads just prior to use:
Transfer 20 μl of bead slurry to a clean tube. Place the tube in a magnetic separation rack for 10-15 seconds.
Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.
IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml-1.0 mg/ml is recommended.
IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, and Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples.
Proceed to one of the following specific set of steps.
NOTE: For proteins with molecular weights in the range of around 50 kDa, we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) as a secondary antibody to minimize interference produced by denatured heavy chains. For proteins with molecular weights in the range of around 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) is recommended to minimize interference produced by denatured light chains.
posted December 2008
revised April 2018
Protocol Id: 121
Protein G Magnetic Beads are an affinity matrix for the small-scale isolation of immunocomplexes from immunoprecipitations (IP assays). Protein G is covalently coupled to a magnetic particle.
Protein G exhibits high affinity for subclasses of IgG from many species (including human, rabbit, mouse, rat, and sheep) and can be used for immunoprecipitation assays with these antibodies. Beads can be separated from solution using our 6-Tube Magnetic Separation Rack #7017 or 12-Tube Magnetic Separation Rack #14654 which concentrates the beads to the side of the tube instead of the bottom. This eliminates centrifugation steps, minimizes sample loss, increases washing efficiency, and saves time.
The 1mL and 5mL size is enough material for 25 and 125 immunoprecipitations, respectively, when following our recommended protocol.
Bead Diameter: ~1.5 μm
Binding Capacity: > 0.2 μg Rabbit IgG/μl bead slurry