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 !Incubate cultured cells (80-90% confluent monolayer in 100 mm cell cultureplate or approximately 2-5 x 107suspension cells in flask).

2 !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 .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.

3 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. 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.

4 !Wash pellet 4 times with ml RIPA buffer (more stringent) or PBS (lessstringent), each time repeating centrifugation step above.!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. Cell 95: , , et al. 2011. E2F1-mediated upregulation of p19 INK4ddetermines its periodic expression during cell cycle and regulates cellularproliferation.

5 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. 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.

6 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. 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.

7 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. 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)

8 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.

9 FIRST AID MEASURES_____First Aid MeasuresGeneral adviceConsult a physician if necessary. Remove to fresh contactWash with plenty of ContactWash skin with soap and to fresh air If breathing is difficult, give oxygen If not breathing, give artificialrespirationIngestionNever give anything by mouth to an unconscious person. Clean mouth with important symptoms and effects, both acute and delayedSymptomsNo information of any immediate medical attention and special treatment neededNote to physiciansTreat FIRE-FIGHTING MEASURES_____Suitable Extinguishing MediaSuitable Extinguishing MediaUse extinguishing measures that are appropriate to local circumstances and thesurrounding Extinguishing hazards arising from the chemicalSpecific hazards arising from thechemicalNo information combustion productsNo information dataSensitivity to Mechanical ImpactNo information to Static DischargeNo information equipment and precautions for firefightersProtective equipment and precautionsfor firefightersAs in any fire.

10 Wear self-contained breathing apparatus pressure-demand, MSHA/NIOSH(approved or equivalent) and full protective ACCIDENTAL RELEASE MEASURES_____Personal precautions, protective equipment and emergency proceduresPersonal precautionsEnsure adequate ventilation, especially in confined precautionsEnvironmental precautionsSee Section 12 for additional Ecological and material for containment and cleaning upMethods for containmentPrevent further leakage or spillage if safe to do for cleaning upPick up and transfer to properly labeled HANDLING AND STORAGES anta cruz Biotechnology, Inc. - Protein A/G plus -Agarose2 / 5 Revision date 11-Dec-2017_____Precautions for safe handlingAdvice on safe handlingHandle in accordance with good industrial hygiene and safety for safe storage, including any incompatibilitiesStorage ConditionsKeep containers tightly closed in a dry, cool and well-ventilated place.