SANTA CRUZ BIOTECHNOLOGY, INC. Western Blotting Luminol Reagent: sc-2048 PRODUCT NOTES Santa Cruz Biotechnology offers Western Blotting Luminol Reagent for en- Ⅲ Have all materials ready before beginning the procedure. The following hanced chemiluminescence detection of Western blots. Luminol should be materials are needed: plastic wrap (enough to cover the blot), scissors, used in conjunction with horseradish peroxidase (HRP)-conjugated primary tweezers, tape, several pieces of film, film cassette, paper backing (size antibodies, HRP-conjugated secondary antibodies or mouse IgG binding pro- of film cassette) and a screw cap vial. teins. One Luminol package contains 125 ml each Solution A and Solution B, Ⅲ Solutions contain chemicals that may bleach skin or clothing. Always which combine to form sufficient reagent for a 2,000 cm2 membrane area. wear gloves, protective clothing and eyewear to avoid solution contact. PROCEDURE Ⅲ Nitrocellulose or PVDF membrane should be handled with tweezers or gloved hands only. Ⅲ Perform Western blot onto nitrocellulose or PVDF membrane according to Santa Cruz Biotechnology Protocols or according to standard protocols. For Ⅲ Work quickly and carefully as maximal detection and light emission due direct ECL detection, we recommend the use of SCBT’s horseradish peroxidase to enzymatic reaction occur during the first hour. (HRP)-conjugated primary antibodies. For indirect detection, probe membrane Ⅲ If high backgrounds are observed following a 1 minute exposure of mem- with primary antibody followed by HRP-conjugated secondary antibody or brane to film, allow membrane to sit in the dark for 20-30 minutes at room mouse IgG binding protein (m-IgGk-BP-HRP: sc-516102, or m-IgGl-BP-HRP: temperature, followed by an additional 1 minute exposure of membrane to sc-516132). For a Cruz Marker™ (sc-2035) compatible mouse IgG kappa binding film. Alternatively, repeat the Western blot with a higher dilution of second- protein, use m-IgGk BP-HRP (Cruz Marker) (sc-516102-CM), or m-IgGl BP-HRP ary antibody or mouse IgG binding proteins (up to 1:10,000). (Cruz Marker) (sc-516132-CM). Ⅲ Wash membrane three times for 5 minutes each with 1x TBS, 0.05% DATA Tween-20. Wash once for 5 minutes with 1x TBS [1x Tris buffered saline (TBS): 50 mM Tris-HCl, pH 7.4; 150 mM NaCl]. ABC D E F 132 K ABC D E F < Daxx 132 K Ⅲ Mix equal quantities of Luminol Reagent Solution A and Solution B by 90 K inversion in a screw cap vial. Use 0.125 ml/cm2 total volume. < CAS 90 K 55 K Ⅲ Pour off TBS wash from the nitrocellulose or PVDF membrane. Add the mixed Luminol Reagent Solutions to the membrane, protein side facing up. 43 K 55 K Incubate for 1 minute at room temperature. 34 K 23 K Ⅲ Pour off excess Luminol Reagent, or lift membrane with tweezers to drain Daxx (M-112): sc-7152. Western blot analysis of Daxx CAS (N-19): sc-1709. Western blot analysis of CAS excess Reagent and lightly dab the edge of the membrane with a chemwipe expression in HeLa (A), BJAB (B), Ramos (C), K-562 (D), expression in NIH/3T3 (A), RAW 264.7 (B) and MM- to remove excess solution. Use caution not to wipe or smear the membrane RAW 264.7 (E) and WEHI-231 (F) whole cell lysates. 142 (C) nuclear extracts and EOC 20 (D), F9 (E) and c4 (F) whole cell lysates. surface. Ⅲ Tightly cover the membrane, protein side facing up, with a single layer of SELECT PRODUCT CITATIONS plastic wrap. Be careful to prevent the formation of wrinkles or bubbles 1. Blumenthal, T., et al. 2000. Degranulating eosinophils in human endome- between the membrane and the plastic wrap. triosis. Am. J. Pathol. 156: 1581-1588. Ⅲ Carefully tape the plastic wrapped membrane, protein side facing up, to 2. Blanco, E., et al. 2012. Attenuation of cocaine-induced conditioned the paper backing. If desired, label membrane by applying phosphorescent locomotion is associated with altered expression of hippocampal glu- tape to the outside of the plastic wrap. Place in the film cassette. tamate receptors in mice lacking LPA1 receptors. Psychopharmacology Ⅲ In a darkroom, expose membrane to film for 1 minute and develop according 220: 27-42. to standard protocols for autoradiogram. Exposure period may be increased as 3. Vega-Naredo, I., et al. 2012. Melatonin modulates autophagy through a necessary. redox-mediated action in female syrian hamster Harderian gland control- ling cell types and gland activity. J. Pineal Res. 52: 80-92. STORAGE 4. Burmeister, D., et al. 2012. Impact of partial urethral obstruction on bladder Luminol Reagent Solutions are stable at room temperature for one hour. function: time-dependent changes and functional correlates of altered Store at 2-8° C for longer periods. expression of Ca2+ signaling regulators. Am. J. Physiol. Renal Physiol. 302: F1517-F1528. RESEARCH USE 5. Moreno-Igoa M., et al. 2012. Non-viral gene delivery of the GDNF, either For research use only, not for use in diagnostic procedures. alone or fused to the C-fragment of tetanus toxin protein, prolongs survival in a mouse ALS model. Restor. Neurol. Neurosci. 30: 69-80. 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 SAFETY DATA SHEET Santa Cruz Biotechnology, Inc. Revision date 02-Dec-2019 Version 2.3 1. IDENTIFICATION OF THE SUBSTANCE/PREPARATION AND OF THE COMPANY/UNDERTAKING _____________________________________________________________________________________________________________________ Product identifier Product Name Western Blotting Luminol Reagent: Solution A Product Code SC-2048 Recommended use of the chemical and restrictions on use For research use only. Not intended for diagnostic or therapeutic use. Details of the supplier of the safety data sheet Emergency telephone number Santa Cruz Biotechnology, Inc. Chemtrec 10410 Finnell Street 1.800.424.9300 (Within USA) Dallas, TX 75220 +1.703.527.3887 (Outside USA) 831.457.3800 800.457.3801 [email protected] 2. HAZARDS IDENTIFICATION _____________________________________________________________________________________________________________________ This chemical is not considered hazardous by the 2012 OSHA Hazard Communication Standard (29 CFR 1910.122). Classification Not a dangerous substance or mixture according to the Globally Harmonized System (GHS) Label elements Signal word Not classified Hazard statements Not classified Symbols/Pictograms Not classified Precautionary Statements - Prevention Wash hands thoroughly after handling Precautionary Statements - Response IF exposed or concerned: Get medical advice/attention Hazards not otherwise classified (HNOC) Hazards not otherwise classified (HNOC) Not applicable Other Information NFPA Health hazards - HMIS Health hazards - Flammability - Flammability - Stability - Physical hazards - Physical and chemical - Personal protection - properties 3. COMPOSITION/INFORMATION ON INGREDIENTS _____________________________________________________________________________________________________________________ Molecular Weight No information available Formula No information available Chemical name CAS No. Weight-% Oral LD50 Dermal LD50 Inhalation LC50 Water 7732-18-5 >95 > 90 mL/kg ( Rat ) - - Dimethyl Sulfoxide (DMSO) 67-68-5 1 - 5 = 28300 mg/kg ( Rat ) = 40 g/kg ( Rat ) - SC-2048 - Western Blotting Luminol Reagent: Solution A Revision date 02-Dec-2019 _____________________________________________________________________________________________________________________ = 14500 mg/kg ( Rat ) Tris base 77-86-1 1 - 5 = 5900 mg/kg ( Rat ) - - Luminol 521-31-3 <0.1 - - - p-Coumaric acid 501-98-4 <0.1 - - - 4. FIRST AID MEASURES _____________________________________________________________________________________________________________________ First Aid Measures General advice Consult a physician if necessary. Remove to fresh air. Eye contact Wash with plenty of water. Skin Contact Wash skin with soap and water. Inhalation Remove to fresh air If breathing is difficult, give oxygen If not breathing, give artificial respiration Ingestion Never give anything by mouth to an unconscious person. Clean mouth with water. Most important symptoms and effects, both acute and delayed Symptoms No information available. Indication of any immediate medical attention and special treatment needed Note to physicians Treat symptomatically. 5. FIRE-FIGHTING MEASURES _____________________________________________________________________________________________________________________ Suitable Extinguishing Media Suitable Extinguishing Media Use extinguishing measures that are appropriate to local circumstances and the surrounding environment. Unsuitable Extinguishing Media No information available. Specific hazards arising from the chemical Specific hazards arising from the No information available. chemical Hazardous combustion products No information available. Explosion data Sensitivity to Mechanical Impact No information available. Sensitivity to Static Discharge No information available. Protective equipment and precautions for firefighters Protective equipment and precautions As in any fire, wear self-contained breathing apparatus pressure-demand, MSHA/NIOSH for firefighters (approved or equivalent) and full protective gear. 6. ACCIDENTAL RELEASE MEASURES _____________________________________________________________________________________________________________________ Personal precautions, protective equipment and emergency procedures Personal precautions Ensure adequate ventilation, especially in confined areas. Environmental precautions Environmental precautions