Download Protease/Phosphatase Inhibitor Cocktail (100X)

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
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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
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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
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