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santacruz G蛋白PLUS-瓊脂糖Protein G PLUS-Agarose

SANTA CRUZ BIOTECHNOLOGY, INC.Protein G PLUS-AgaroseImmunoprecipitation Reagent: sc-2002Santa Cruz Biotechnology, Inc. 1.800.457.3801 831.457.3800 831.457.3801 Europe +00800 4573 8000 49 6221 4503 0 www.scbt.comPRODUCTProtein G PLUS is provided as an agarose conjugate for use in immunoprecipitationonly. The product is provided as 0.5 ml agarose in 2.0 ml PBS bufferwith 0.02% azide. Protein G PLUS-Agarose is pre-blocked with BSA to reducenon-specific immunoglobulin binding. Sufficient product is provided for 100immunoprecipitation reactions, to be used at 20 μl resuspended volume perreaction.SPECIFICITYProtein G PLUS-Agarose is suitable for immunoprecipitation of mouse IgG1,IgG2a, IgG2b and IgG3, rat IgG1, IgG2a, IgG2b and IgG2c, rabbit and goat polyclonalAbs, and human IgG1, IgG2, IgG3 and IgG4.PROCEDURE? Incubate cultured cells （80-90% confluent monolayer in 100 mm cell cultureplate, or approximay 2-5 x 107 suspension cells in flask） in methioninefreemedium containing 5% dialyzed fetal calf serum for 1 hour at 37° C.The same procedure can be used for cells labeled with other radioactiveamino acids （e.g., 14C or 3H） or with γ32P-orthophosphate. Cell labeling mustbe carried out in medium lacking the relevant amino acid or in phosphatefreemedium.? Remove medium and replace with 3 ml methionine-free medium containing5% dialyzed fetal calf serum and 100 μCi/ml 35S-methionine. Incubate 1hour 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 cellmonolayer with PBS.? Add 3 ml ice cold RIPA buffer to cell monolayer and incubate at 4° C for10 minutes. For suspension cells, add the RIPA buffer to washed cell pelletin a 15 ml conical centrifuge tube.? Disrupt cells by repeated aspiration through a 21 gauge needle and transferto a 15 ml conical centrifuge tube.? Wash cell culture plate with additional 1.0 ml ice cold RIPA buffer andcombine with original extract.? Pellet cellular debris by centrifugation at 10,000 xg 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 controlIgG （normal mouse, rat, rabbit or goat IgG, corresponding to the hostspecies of the primary antibody）, together with 20 μl of resuspendedvolume of Protein G PLUS-Agarose. Incubate at 4° C for 30 minutes.? Pellet beads by centrifugation at 2,500 rpm （approximay 1,000 xg） for5 minutes at 4° C. Transfer supernatant （cell lysate） to a fresh 15 ml conicalcentrifuge tube on ice.? Transfer 1 ml of the above cell lysate, or approximay 100-500 μg totalcellular 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 bytitration） and incubate for 1 hour at 4° C.? Add 20 μl of resuspended volume of Protein G PLUS-Agarose. Cap tubesand incubate at 4° C on a rocker platform or rotating device for 1 hour toovernight.? Collect immunoprecipitates by centrifugation at 2,500 rpm （approximay1,000 xg） for 5 minutes at 4° C. Carefully aspirate and discard radioactivesupernatant.? Wash pellet 4 times with 1.0 ml RIPA buffer （more stringent） or PBS （lessstringent）, each time repeating centrifugation step above.? After final wash, aspirate and discard supernatant and resuspend pellet in40 μl of 1x electrophoresis sample buffer.? Boil samples for 2–3 minutes and analyze 20 μl aliquots by SDS-PAGE andautoradiography. Unused samples may be stored at -20° C.? Optional: After boiling, samples may be centrifuged to pellet the agarosebeads followed by SDS-PAGE analysis of the supernatant.SELECT PRODUCT CITATIONS1. Medema, R., et al. 1998. p21waf1 can block cells at two points in the cellcycle, but does not interfere with processive DNA-replication or stressactivatedkinases. Oncogene 16: 431-441.2. Marzo, I., et al. 1998. Bax and adenine nucleotide translocator cooperatein the mitochondrial control of apoptosis. Science 281: 2027-2031.3. Song, Y., et al. 2011. Ligand-dependent corepressor acts as a novel corepressorof thyroid hormone receptor and represses hepatic lipogenesis inmice. J. Hepatol. 56: 248-254.4. Beard, R.S., et al. 2012. Metabotropic glutamate receptor 5 mediatesphosphorylation of vascular endothelial cadherin and nuclear localizationof β-catenin in response to homocysteine. Vascul. Pharmacol. 56: 159-167.STORAGEStore at 4° C, do not freeze; stable for one year from the date of shipment.RESEARCH USEFor research use only, not for use in diagnostic procedures.Immunoprecipitation agarose conjugates are pre-blocked with BSA to reduce non-specific immunoglobulinbinding and are provided at a concentration （0.5 ml agarose/2.0 ml） suitable for use at 20 μl perimmunoprecipitation reaction. Number of reactions: 100.

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