SANTA CRUZ BIOTECHNOLOGY, INC. Protein A/G PLUS …

1 SANTA cruz BIOTECHNOLOGY, A/G PLUS-Agarose: sc-2003 SANTA cruz Biotechnology, fax Europe+00800 4573 8000 49 6221 4503 0 A/G PLUS is provided as an agarose conjugate for use in immunopre-cipitation only. The product is provided as ml agarose in ml PBS bufferwith azide. Protein A/G PLUS-Agarose is pre-blocked with BSA toreduce non-specific immunoglobulin binding. Sufficient product is provided for100 immunoprecipitation reactions, to be used at 20 l resuspended volumeper A/G PLUS-Agarose is suitable for immunoprecipitation of mouseIgG1, IgG2a, IgG2b, IgG3and IgA, rat IgG1, IgG2a, IgG2band IgG2c, rabbit andgoat polyclonal Abs, and human IgG1, IgG2, IgG3and !

2 Incubate cultured cells (80-90% confluent monolayer in 100 mm cell cultureplate or approximately 2-5 x 107suspension cells in flask).!Add 3 ml ice cold RIPA buffer to cell monolayer and incubate at 4 C for10minutes. 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 ml ice cold RIPA buffer andcombine with original extract.!Pellet cellular debris by centrifu gation at 10,000 xg for 10 minutes at 4 a n s f e r s u p e r n a t a n t t o a f r e s h 1 5 m l c o n i c a l c e n t r i f u g e t u b e o n i c e.

3 Preclear lysate (optional step) by adding g of the appropriate controlIgG (normal mouse, rat, rabbit or goat IgG, corresponding to the hostspecies of the primary antibody), together with 20 l of resuspended volume of Protein A/G PLUS-Agarose. Incubate at 4 C for 30 minutes.!Pellet beads by centrifu gation at 2,500 rpm (approximately 1,000 xg) for 5 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 approximately 100-500 g totalcellular Protein , to a ml microcentrifuge tube.

4 Add 1-10 l ( , g)primary antibody (optimal antibody concentration should be determined bytitration) and incubate for 1 hour at 4 C.!Add 20 l of resuspended volume of Protein A/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 2,500rpm (approx imately1,000 xg) for 5minutes at 4 C. Carefully aspirate and discard supernatant.!Wash pellet 4 times with ml RIPA buffer (more stringent) or PBS (lessstringent), each time repeating centrifugation step above.

5 !After final wash, aspirate and discard supernatant and resuspend pelletin 40 l of 1x electrophoresis sample buffer.!Boil samples for 2-3 minutes and analyze 20 l aliquots by SDS-PAGEand autoradiography. 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 PRODUCT , and Zhu, Q. 1994. Signaling by ATP receptors in 5: , M., et al. 1998. PIF3, a phytochrome-interacting factor necessary fornormal photoinduced signal transduction, is a novel basic helix-loop-helixprotein.

6 Cell 95: , , et al. 2011. E2F1-mediated upregulation of p19 INK4ddetermines its periodic expression during cell cycle and regulates cellularproliferation. PLoS ONE 6: , , et al. 2011. Molecular mechanisms of activation of endothelialnitric oxide synthase mediated by transient receptor potential vanilloidtype 1. Cardiovasc. Res. 91: , , et al. 2012. N-terminal domain of soluble epoxide hydrolasenegatively regulates the VEGF-mediated activation of endothelial nitricoxide synthase. Cardiovasc. Res. 93: DP,, et al. 2013.

7 Identification and characterization of PlAlix,the Alix homologue from the Mediterranean sea urchin Paracentrotuslividus. Dev Growth Differ. 55: D,, et al. 2013. Generation and nuclear translocation of sumoylatedtransmembrane fragment of cell adhesion molecule L1. J Biol Chem. 287 J,, et al. 2013. A noncatalytic function of the ligation complexduring nonhomologous end joining. J. Cell Biol. 200: at 4 C, do not freeze; stable for one year from the date of USEFor research use only, not for use in diagnostic agarose conjugates are pre-blocked with BSA to reduce non-specific immunoglobulinbinding and are provided at a concentration ( ml ml) suitable for use at 20 l perimmunoprecipitation reaction.

8 Number of reactions: #AMOUNTIMMUNOPRECIPITATION REAGENTSP rotein A-Agarosemouse IgG2a, IgG2band mlrabbit polyclonal Abshuman IgG1, IgG2and IgG4 Protein G PLUS-Agarosemouse IgG1, IgG2a, IgG2band mlrat IgG1, IgG2a, IgG2band IgG2crabbit and goat polyclonal Abshuman IgG1, IgG2, IgG3and IgG4 Protein A/G PLUS-Agaroseall of the above mlProtein L-Agarosemouse, rat, human IgG, scFv and mlfragments, mouse and human IgM, IgE and IgA1. IDENTIFICATION OF THE SUBSTANCE/PREPARATION AND OF THE COMPANY/UNDERTAKING_____Product identifierProduct NameProtein A/G PLUS-AgaroseProduct CodeSC-2003 Recommended use of the chemical and restrictions on useFor research use only.

9 Not intended for diagnostic or therapeutic HAZARDS IDENTIFICATION_____This chemical is not considered hazardous by the 2012 OSHA Hazard Communication Standard (29 CFR ).ClassificationNot a dangerous substance or mixture according to the Globally Harmonized System (GHS)Label elementsSignal wordNot classifiedPrecautionary Statements - PreventionWash hands thoroughly after handlingHazards not otherwise classified (HNOC)Hazards not otherwise classified (HNOC)Not applicableOther InformationUnknown acute toxicity100% of the mixture consists of ingredient(s)

10 Of unknown COMPOSITION/INFORMATION ON INGREDIENTS_____Molecular WeightNo information availableFormulaNo information availableChemical NameCAS NoWeight %Oral LD50 Dermal LD50 Inhalation LC50 PBS with Azide-70 - 90---HMISP recautionary Statements - ResponseHazard statementsDetails of the supplier of the safety data sheetSanta cruz Biotechnology, Finnell StreetDallas, TX classifiedEmergency telephone (Within USA)+ (Outside USA)NFPAS anta cruz Biotechnology, Inc. / 5 Health hazards-IF exposed or concerned: Get medical advice/attentionHealth hazards-Flammability-Flammability-SAFETY DATA SHEETS anta cruz Biotechnology, hazards-Stability-Personal protection-Not classifiedPhysical and chemicalproperties-Version 1 Revision date 11-Dec-2017_____Protein G PLUS-Agarose-7 - 13--- Protein A-Agarose-7 - 13---4.