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Comment
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Upon initial use of this product, we recommend that the vial be inverted several times to get the beads into suspension. We recommend to use a large bore pipet to pipet up the liquid for use. For storage of the opened vial, we recommend that the vial cap be sealed with parafilm to help prevent evaporation of the buffer.
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Assay Procedure
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Preparation of Immunoprecipitated Sample for SDS-PAGE:
1. Preclear cell lysate: Add 50 μL of Anti-Mouse IgG Beads and 500μl of cell lysate sample to a microcentrifuge tube and incubate on ice for
3. minutes. Spin at 10,000xg for 3 minutes and transfer the supernatant to a new microcentrifuge tube.
2. Immunoprecipitation: Add 5 μg of primary antibody to the microcentrifuge tube containing the precleared lysate. Incubate on ice for 1 hour. Add 50 μL of Anti-Mouse IgG Beads. Incubate for 1 hour on a rocking platform. Spin the microcentrifuge tube at 10000xg for 1 minute. Remove supernatant completely and wash the (pelleted) beads 3 times with 500 μL of Lysis Buffer (50mM Tris HCl pH 8.0, 150mM NaCl,1 % NP-40).
3. Prepare sample for SDS-PAGE: After the last wash, aspirate supernatant, and add 100 μL Laemmli Buffer (with 50 mM DTT or 1 %?-mercaptoethanol, final) to bead pellet. Vortex and heat to 90-100 °C for
1. minutes. Spin at 10000xg for 3 minutes, collect supernatant, and load onto the gel. Avoid loading Anti-Mouse Ig Beads.
Note: The supernatant can be stored at -20 °C for future use. After thawing, add fresh dithiothreitol and heat as above. Centrifuge the sample at 10000xg for 1 minute in a microcentrifuge tube to pellet any Anti-Mouse Ig Beads and immediately transfer an aliquot of the supernatant to gel wells.
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