TrueBlot® Anti-Rabbit Ig IP Agarose Bead from Rockland Immunochemicals, Inc.

Application Note: Upon initial use of this product, we recommend that the vial be inverted several times to get the beads into suspension. We recommend using 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. Procedure: Preparation of Immunoprecipitated Sample for SDS-PAGE: 1. Preclear cell lysate: Add 50 µL of Anti-Rabbit IgG Beads and 500µl of cell lysate sample to a microcentrifuge tube and incubate on ice for 30 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-Rabbit IgG Beads. Incubate for 1 hour on a rocking platform. Spin the microcentrifuge tube at 10,000xg 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 50 µL Laemmli Buffer (with 50 mM DTT or 2% β-mercaptoethanol, final) to bead pellet. Vortex and heat to 90-100 °C for 10 minutes. Spin at 10,000xg for 3 minutes, collect supernatant, and load onto the gel. Avoid loading Anti-Rabbit 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 10,000xg for 1 minute in a microcentrifuge tube to pellet any Anti-Rabbit Ig Beads and immediately transfer an aliquot of the supernatant to gel wells.