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Store at 4ºC Protease/Phosphatase Inhibitor Cocktail (100X) www.cellsignal.com Support: 877-678-TECH (8324) www.cellsignal.com/support #5872 1 ml Orders: 877-616-CELL (2355) [email protected] rev. 02/04/16 For Research Use Only. Not For Use In Diagnostic Procedures. Description: When diluted in lysis buffer to a final concentration of 1X the Protease/Phosphatase Inhibitor Cocktail prevents protein degradation and dephosphorylation by endogenous proteases and phosphatases present in the whole cell extract. The 100X cocktail is a clear light yellow to light green liquid. Background: Dynamic protein phosphorylation is a key cellular signaling mechanism by which a broad spectrum of cellular processes is regulated. In order to study the phosphorylation status of specific target proteins the phosphorylated residue of interest must remain intact. When cells are lysed to make whole cell extracts, a loss of normal cellular signaling regulation occurs, and phosphatases within the cell extract are free to dephosphorylate proteins in an uncontrolled manner. The addition of phosphatase inhibitors to the cell lysis buffer aids in the preservation of phosphorylated residues at the time of cell disruption. This same loss of normal cellular control when generating whole cell extracts also leads to uncontrolled degradation of proteins by endogenous proteases. The addition of protease inhibitors to the cell lysis buffer aids in the preservation of target proteins in the cell extract. Directions for Use: 1. Briefly vortex the Protease/Phosphatase Inhibitor Cocktail (100X) before use. For product specific protocols and a complete listing of recommended companion products please see the product web page at www.cellsignal.com 2. Just prior to lysing cells, dilute the cocktail 1:100 in desired lysis buffer to obtain a 1X working concentration. Solutions and Reagents: The Protease/Phosphatase Inhibitor Cocktail (100X) is composed of a proprietary mix of Aprotinin, Bestatin, E64, and Leupeptin to promote broad spectrum protection against endogenous proteases and sodium fluoride, sodium pyrophosphate, β-glycerophosphate, and sodium orthovanadate to promote broad spectrum protection against endogenous serine/threonine and tyrosine phosphatases. The cocktail does not contain EDTA (a metalloprotease inhibitor) which can be incompatible with some downstream applications (i.e. protein assays, 2D electrophoresis, etc.). If EDTA is desired as a protease inhibitor it can be added to the cell lysis buffer at a final working concentration of 5mM. kDa kDa Storage: Store the undiluted 100X cocktail at 4ºC. Do not freeze. This product is stable for 12 months. no protease inhibitors #5872 added 200 no phosphatase inhibitors #5872 added 140 200 140 100 100 80 β-Catenin 80 degradation product Phospho-Akt (Ser 473) 60 50 60 50 40 0 60 3 6 20 0 3 6 20 hrs Akt 50 0 3 6 20 0 3 6 20 hrs Western blot analysis of extracts from NIH/3T3 cells, serum-starved overnight and treated with hPDGF-BB #8912 (100ng/ml, 5min), prepared in lysis buffer in the absence of phosphatase inhibitors (left) or with Protease/Phosphatase Inhibitor Cocktail (100X) #5872 added (right), and incubated at 37ºC for the indicated time points, using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower). In the absence of phophatase inhibitors, phospho-Akt signal drops significantly after time point 0, demonstrating rapid loss of phosphorylation at later time points. In the presence of the phosphatase inhibitor cocktail, the phospho-Akt signal is preserved through all time points monitored. Western blot analysis of extracts from NIH/3T3 cells, prepared in lysis buffer in the absence of protease inhibitors (left) or with Protease/Phosphatase Inhibitor Cocktail (100X) #5872 added (right), and incubated at 37ºC for the indicated time points, using β-Catenin (D10A8) XP® Rabbit mAb #8480. In the absence of protease inhibitors, β-Catenin signal fades within 3 hr after harvest, indicating protein degradation. In the presence of the protease inhibitor cocktail, the β-Catenin degradation is slowed significantly and signal is still present at 20 hr following harvest. Thank you for your recent purchase. If you would like to provide a review visit www.cellsignal.com/comments. © 2014 Cell Signaling Technology, Inc. XP® and Cell Signaling Technology® are trademarks of Cell Signaling Technology, Inc. Applications: W—Western IP—Immunoprecipitation IHC—Immunohistochemistry ChIP—Chromatin Immunoprecipitation IF—Immunofluorescence F—Flow cytometry E-P—ELISA-Peptide Species Cross-Reactivity: H—human M—mouse R—rat Hm—hamster Mk—monkey Mi—mink C—chicken Dm—D. melanogaster X—Xenopus Z—zebrafish B—bovine Dg—dog Pg—pig Sc—S. cerevisiae Ce—C. elegans Hr—Horse All—all species expected Species enclosed in parentheses are predicted to react based on 100% homology. Material Safety Data Sheet (MSDS) Material Safety Data Sheet (MSDS) SDS# : 5872 Revision Date 2012-06-04 Version 2 SDS# : 5872 Revision Date 2012-06-04 Version 2 ______________________________________________________________________________________ 1. PRODUCT AND COMPANY IDENTIFICATION Product Name Product Number Identified Uses Protease/Phosphatase Inhibitor Cocktail (100X) 5872 For Research Use Only (RUO). Not intended for use in humans or animals. Not intended for therapeutic or diagnostic procedures. Manufacturer/Supplier Cell Signaling Technology, Inc. 3 Trask Lane Danvers, MA 01923 USA 1-978-867-2300 1-978-578-6737 Phone #: Emergency Tel# Emergency Overview Target organ effects Concentration 819-83-0 7758-16-9 7681-49-4 13721-39-6 9087-70-1 3-5% 1-3% 1-3% 1-3% 0.1-1% Eye Contact Skin Contact Inhalation Ingestion Rinse immediately with plenty of water. Get medical attention. Rinse immediately with soap and plenty of water. Get medical attention. Move to fresh air. Get medical attention. Call Poison Control Center immediately. Never give anything by mouth to an unconscious person. Rinse mouth with water. Treat symptomatically. _____________________________________________________________________________________________ 5. FIRE FIGHTING MEASURES Flash Point See Section 9. Physical and Chemical Properties Suitable Extinguishing Media Use dry chemical Special exposure hazards No data available Special protection for fire fighters Wear appropriate self-contained breathing apparatus and protective unit Other information No data available _____________________________________________________________________________________________ 6. ACCIDENTAL RELEASE MEASURES Physical State Liquid Mutagenic effects Teratogenic effects Reproductive toxicity Sensitization CAS No. Disodium beta-glycerophosphate Disodium dihydrogenpyrophosphate Sodium fluoride Trisodium tetraoxovanadate Tyrpsin inhibitor, pancreatic basic Notes to physician Warning! This material is considered hazardous by the OSHA Hazard Communication Standard (29 CFR 1910.1200) Harmful if inhaled or swallowed. Causes respiratory tract, eye, and skin irritation. May be harmful if absorbed through skin. Contains material that can cause target organ damage. Suspect cancer hazard – contains material which may cause cancer. Potential Health Effects Acute Toxicity Eyes Skin Inhalation Ingestion Chronic Effects Carcinogenic effects Chemical Name _____________________________________________________________________________________________ 4. FIRST AID MEASURES _____________________________________________________________________________________________ 2. HAZARDS IDENTIFICATION Routes of Entry: Hazardous Ingredients Dermal contact. Eye contact. Inhalation. Ingestion. Personal precautions Do not touch or walk though spilled material. Wear personal protective equipment Do not let product enter drains. Soak up with absorbent material. Keep in suitable closed containers for disposal. Other information See sections 12 and 13 for additional information. ____________________________________________________________________________________________ 7. HANDLING AND STORAGE Environmental precautions Clean up methods Irritating to eyes. Harmful if contact with skin Toxic if inhaled. Irritating to respiratory system. Toxic if swallowed Contains material which may cause cancer. Risk of cancer depends on duration and level of exposure. No known significant effects or critical hazards. No known significant effects or critical hazards. No known significant effects or critical hazards. No information available Contains material which causes damage to the following organs: Heart. Teeth. Contains material which causes damage to the following organs: Kidneys, gastrointestinal tract, upper respiratory tract, skin, bones, central nervous system (CNS), eye, lens, or cornea. _____________________________________________________________________________________________ 3. COMPOSITION/INFORMATION ON INGREDIENTS 1 Cell Signaling Technology, Inc www.cellsignal.com Safe handling advice Avoid contact with eyes and skin. Ensure adequate ventilation. Storage conditions Keep container tightly closed in a cool dry location. Incompatibilities No data available Specific end uses No data available _____________________________________________________________________________________________ 8. EXPOSURE CONTROLS / PERSONAL PROTECTION Exposure Limits Chemical Name Sodium fluoride Ingredients with workplace control parameters OSHA PEL (TWA) OSHA PEL (Z2) 3 ACGIH OEL (TLV) 3 2.5 mg/m 8hours. (as F) Exposure Controls Apply technical measures to comply with the occupational exposure limits. Engineering Controls Emergency eyewash and safety shower. Mechanical exhaust required. Hygiene Measures Do not eat, drink or smoke when handling product. Wash hands thoroughly after handling product. Wash contaminated clothing before reuse. Personal Protective Equipment Respiratory Protection: Eye Protection: Skin and body protection: Hand protection: 2 Material Safety Data Sheet (MSDS) Information on basic physical and chemical properties Physical State Appearance Odor pH Water Solubility Melting point/freezing point Boiling point Evaporation rate Flash point Autoignition temperature Flammability limits in air Explosive properties VOC content Decomposition temperature liquid no data available odorless no data available Soluble no data available no data available no data available no data available no data available no data available no data available no data available no data available SDS# : 5872 Revision Date 2012-06-04 Version 2 Risk of serious damage to eyes Toxic in contact with skin Toxic by inhalation Toxic if swallowed Ingredient Name Result Species Dose Disodium dihydrogenpyrophosphate Sodium fluoride LC50 Inhalation Dust and Mist LD50 Oral LD50 Oral Eyes Moderate Irritant LD50 Oral Rat Rat Rat Rabbit Rat >0.58mg/L 1800 mg/kg 31 mg/kg Mutagenic effects Teratogenic effects Reproductive toxicity Sensitization 330/mg/kg Contains material which may cause cancer. Risk of cancer depends on duration and level of exposure. No known significant effects or critical hazards. No known significant effects or critical hazards. No known significant effects or critical hazards. No information available Ingredient Name ACGIH IARC EPA NIOSH NTP OSHA Sodium fluoride Trisodium tetraoxovanadate A4 - 3 - C - None Possible - None Target organ effects Contains material which causes damage to the following organs: Heart. Teeth. Contains material which causes damage to the following organs: Kidneys, gastrointestinal tract, upper respiratory tract, skin, bones, central nervous system (CNS), eye, lens, or cornea. _____________________________________________________________________________________________ 12. ECOLOGICAL INFORMATION _____________________________________________________________________________________________ 10. STABILITY AND REACTIVITY Stability Conditions/Materials to avoid Hazardous decomposition products Acute Toxicity Eyes Skin Inhalation Ingestion Chronic Effects Carcinogenic effects _____________________________________________________________________________________________ 9. PHYSICAL AND CHEMICAL PROPERTIES 2.5 mg/m 10 hours. (as F) Cell Signaling Technology, Inc www.cellsignal.com Trisodium tetraoxovanadate In case of insufficient ventilation wear suitable respiratory equipment. Safety glasses with side shields Wear suitable protective clothing, protective shoes or boots. Compatible chemical resistant gloves. 3 2.5 mg/m 8hours. (as F) Material Safety Data Sheet (MSDS) SDS# : 5872 Revision Date 2012-06-04 Version 2 NIOSH REL 3 2.5 mg/m 8hours. Form: Dust stable under normal conditions no data available no data available _____________________________________________________________________________________________ 11. TOXICOLOGICAL INFORMATION Ecotoxicity The environmental impact of this product has not been fully investigated. No known significant effects or critical hazards. Persistence and degradability Not available Bioaccumulation Not available Mobility Not available _____________________________________________________________________________________________ 13. DISPOSAL CONSIDERATIONS Waste Disposal Methods To the best of our knowledge, the chemical, physical and toxicological properties have not been fully investigated. Dispose of in accordance with all applicable environmental laws and regulations. _____________________________________________________________________________________________ 14. TRANSPORT INFORMATION Routes of Exposure IATA Not regulated as dangerous goods Potential Health Effects DOT Not regulated as dangerous goods Acute Toxicity Cell Signaling Technology, Inc www.cellsignal.com 3 Cell Signaling Technology, Inc www.cellsignal.com 4 Material Safety Data Sheet (MSDS) Material Safety Data Sheet (MSDS) MEX Not regulated as dangerous goods SDS# : 5872 Revision Date 2012-06-04 Version 2 _____________________________________________________________________________________________ 15. REGULATORY INFORMATION HCS Classification Toxic material Irritating material Carcinogen Target Organ Effects CEPA Toxic Substances Canada Inventory The following components are listed: Inorganic fluorides. At least one component is listed. SDS# : 5872 Revision Date 2012-06-04 Version 2 This product has been classified according to the hazard criteria of the CPR and the MSDS contains all of the information required by the CPR. ____________________________________________________________________________________________ 16. OTHER INFORMATION Revision date Revision Note International Inventories TSCA (8a) PAIR Trisodium tetraoxovanadate EINECS/ELINCS ENCS IECSC KECL PICCS AICS NZIoC - 2012-05-22 ***Indicates updated section Disclaimer The information provided on this material safety data sheet is to the best of our knowledge, information and belief at the date of its publication. The information given is designed only as a guide for safe handling, use, processing, storage, transportation, disposal and release and is not to be considered as a warranty or quality specification. The information relates only to the specific material designated and may not be valid for such material used in combination with any other material or in any process, unless specified in the text. End of Material Safety Data Sheet U.S. Federal Regulations SARA 302/304/311/312 extremely hazardous products: No products were found. SARA 302/304/311/312 emergency planning and notification: No products were found. SARA 302/304/311/312 hazardous chemicals: Sodium fluoride. Trisodium tetraoxovanadate SARA 311/312 MSDS Distribution: Sodium fluoride: Immediate (acute) health hazard, Delayed (chronic) health hazard. Trisodium tetraoxovanadate: Delayed (chronic) health hazard. Commerce Control List precursor: Sodium fluoride Clean Water Act This product the following components are listed as pollutants pursuant to the Clean Water Act (40 CFR 122.21 and 40 CFR 122.42): Sodium fluoride. Clean Air Act, Section 112 Hazardous Air Pollutants (HAPs) (see 40 CFR 61) This product does not contain any substances regulated as hazardous air pollutants (HAPs) under Section 112 of the Clean Air Act Amendments of 1990. U.S. State Regulations Massachusetts New York New Jersey Pennsylvania U.S. Inventory (TSCA 8b) The following components are listed: Sodium fluoride. The following components are listed: Sodium fluoride. The following components are listed: Sodium fluoride. The following components are listed: Sodium fluoride. At least one component is listed. Canada WHMIS Hazard Class D1B Toxic. Material causing immediate and serious toxic effects. D2A Very toxic. Materials causing other toxic effects. D2B Toxic. Materials causing other toxic effects. Canadian NPRI Cell Signaling Technology, Inc www.cellsignal.com The following components are listed: Sodium fluoride.Vanadium. 5 Cell Signaling Technology, Inc www.cellsignal.com 6