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SANTA CRUZ BIOTECHNOLOGY, INC. Protein G PLUS-Agarose Immunoprecipitation Reagent: sc-2002 Santa Cruz Biotechnology, Inc. 1.800.457.3801 831.457.3800 fax 831.457.3801 Europe +00800 4573 8000 49 6221 4503 0 www.scbt.com PRODUCT Protein G PLUS is provided as an agarose conjugate for use in immunoprecip- itation only. The product is provided as 0.5 ml agarose in 2.0 ml PBS buffer with 0.02% azide. Protein G PLUS-Agarose is pre-blocked with BSA to reduce non-specific immunoglobulin binding. Sufficient product is provided for 100 immunoprecipitation reactions, to be used at 20 µl resuspended volume per reaction. SPECIFICITY Protein G PLUS-Agarose is suitable for immunoprecipitation of mouse IgG1, IgG2a, IgG2b and IgG3, rat IgG1, IgG2a, IgG2b and IgG2c, rabbit and goat poly- clonal Abs, and human IgG1, IgG2, IgG3 and IgG4. PROCEDURE • Incubate cultured cells (80-90% confluent monolayer in 100 mm cell culture plate, or approximately 2-5 x 107 suspension cells in flask) in methionine- free medium containing 5% dialyzed fetal calf serum for 1 hour at 37° C. The same procedure can be used for cells labeled with other radioactive amino acids (e.g., 14C or 3H) or with γ32P-orthophosphate. Cell labeling must be carried out in medium lacking the relevant amino acid or in phosphate- free medium. • Remove medium and replace with 3 ml methionine-free medium containing 5% dialyzed fetal calf serum and 100 µCi/ml 35S-methionine. Incubate 1 hour at 37° C. For some proteins a longer labeling period (up to 18 hours) is preferable. • Carefully remove radioactive medium with Pasteur pipette and wash cell monolayer with PBS. • Add 3 ml ice cold RIPA buffer to cell monolayer and incubate at 4° C for 10 minutes. For suspension cells, add the RIPA buffer to washed cell pellet in a 15 ml conical centrifuge tube. • Disrupt cells by repeated aspiration through a 21 gauge needle and transfer to a 15 ml conical centrifuge tube. • Wash cell culture plate with additional 1.0 ml ice cold RIPA buffer and combine with original extract. • Pellet cellular debris by centrifugation at 10,000xg for 10 minutes at 4° C. Transfer supernatant to a fresh 15 ml conical centrifuge tube on ice. Preclear lysate (optional step) by adding 1.0 µg of the appropriate control IgG (normal mouse, rat, rabbit or goat IgG, corresponding to the host species of the primary antibody), together with 20 µl of resuspended volume of Protein G PLUS-Agarose. Incubate at 4° C for 30 minutes. • Pellet beads by centrifugation at 2,500 rpm (approximately 1,000xg) for 5 minutes at 4° C. Transfer supernatant (cell lysate) to a fresh 15 ml conical centrifuge tube on ice. • Transfer 1 ml of the above cell lysate, or approximately 100-500 µg total cellular protein, to a 1.5 ml microcentrifuge tube. Add 1-10 µl (i.e., 0.2-2 µg) primary antibody (optimal antibody concentration should be determined by titration) and incubate for 1 hour at 4° C. • Add 20 µl of resuspended volume of Protein G PLUS-Agarose. Cap tubes and incubate at 4° C on a rocker platform or rotating device for 1 hour to overnight. • Collect immunoprecipitates by centrifugation at 2,500 rpm (approximately 1,000xg) for 5 minutes at 4° C. Carefully aspirate and discard radioactive supernatant. • Wash pellet 4 times with 1.0 ml RIPA buffer (more stringent) or PBS (less stringent), each time repeating centrifugation step above. • After final wash, aspirate and discard supernatant and resuspend pellet in 40 µl of 1x electrophoresis sample buffer. • Boil samples for 2–3 minutes and analyze 20 µl aliquots by SDS-PAGE and autoradiography. Unused samples may be stored at -20° C. • Optional: After boiling, samples may be centrifuged to pellet the agarose beads followed by SDS-PAGE analysis of the supernatant. SELECT PRODUCT CITATIONS 1. Medema, R., et al. 1998. p21waf1 can block cells at two points in the cell cycle, but does not interfere with processive DNA-replication or stress- activated kinases. Oncogene 16: 431-441. 2. Marzo, I., et al. 1998. Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281: 2027-2031. 3. Beard, R.S., et al. 2011. Hyperhomocysteinemia increases permeability of the blood-brain barrier by NMDA receptor-dependent regulation of adherens and tight junctions. Blood 118: 2007-2014. 4. Song, Y., et al. 2011. Ligand-dependent corepressor acts as a novel core- pressor of thyroid hormone receptor and represses hepatic lipogenesis in mice. J. Hepatol. 56: 248-254. 5. Beard, R.S., et al. 2012. Metabotropic glutamate receptor 5 mediates phosphorylation of vascular endothelial cadherin and nuclear localization of β-catenin in response to homocysteine. Vascul. Pharmacol. 56: 159-167. STORAGE Store at 4° C, do not freeze; stable for one year from the date of shipment. RESEARCH USE For research use only, not for use in diagnostic procedures. Immunoprecipitation agarose conjugates are pre-blocked with BSA to reduce non-specific immunoglobulin binding and are provided at a concentration (0.5 ml agarose/2.0 ml) suitable for use at 20 µl per immunoprecipitation reaction. Number of reactions: 100. PRODUCT SPECIFICITY CAT. # AMOUNT IMMUNOPRECIPITATION REAGENTS Protein A-Agarose mouse IgG2a, IgG2b and IgA sc-2001 2.0 ml rabbit polyclonal Abs human IgG1, IgG2 and IgG4 Protein G PLUS-Agarose mouse IgG1, IgG2a, IgG2b and IgG3 sc-2002 2.0 ml rat IgG1, IgG2a, IgG2b and IgG2c rabbit and goat polyclonal Abs human IgG1, IgG2, IgG3 and IgG4 Protein A/G PLUS-Agarose all of the above Abs sc-2003 2.0 ml Protein L-Agarose mouse, rat, human IgG, scFv and Fab sc-2336 2.0 ml fragments, mouse and human IgM, IgE and IgA Administrator 高亮