Download EMD Millipore Protease and Phosphatase Inhibitor Cocktails

Protease and Phosphatase
Inhibitor Cocktails
Pre-mixed formulations to preserve
the integrity of your protein sample
EMD Millipore is a division of Merck KGaA, Darmstadt, Germany
Protease and Phosphatase
Inhibitor Cocktails
Introduction
Ensure the integrity and modification state of
EMD Millipore’s protease and phosphatase inhibitor
purified proteins by using protease and phosphatase
cocktails are provided as ready-to-use, no-waste
inhibitor cocktails, highly specific protease inhibitors
liquid stock solutions with complete formulation
and PhosphoSafe™ Extraction Reagent in cell lysates
details enclosed. Choose the product that’s designed
and tissue extracts. Protease and phosphatase
for your specific application and experience better
inhibitors are essential for successful protein
protein research results.
expression and characterization, biomarker discovery,
mapping of post-translational modifications, protein
expression profiling, protein quantification, and
proteomics research.
Table of Contents
Protease 03
page
Inhibitor Cocktails
08 Phosphatase
page
Inhibitor Cocktails
2
Protease Inhibitor Cocktails
Proper functioning of the cell requires a carefully controlled level of important structural, functional,
and regulatory proteins. Hence, proteins are continuously synthesized and degraded in plant and animal
cells and a finely tuned balance exists between their rate of synthesis and breakdown that determines
the concentration of any given protein. Protein degradation is an essential process whereby damaged,
unwanted, or “used” proteins are continuously eliminated. The extraordinary complexities of proteins in
biological samples present a difficult challenge for their analysis. Hence, in a laboratory set up where the
integrity of proteins is to be maintained during extraction, purification, and analysis the use of protease
inhibitors is highly desired to minimize any non-specific protein breakdown.
Protease inhibitor cocktails are important tools for scientists engaged in a variety of proteomics studies,
including protein characterization, biomarker discovery, mapping of post-translational modifications,
protein expression profiling, and the quantitative measurement of proteins. The addition of a protease
inhibitor cocktail or protease inhibitor ensures the integrity of proteins for downstream analysis and
further characterization of samples.
Advantages
• Convenient: Ready-to-use, waste-free liquid
formulations ensure greater convenience
• Consistent: Stringent quality control ensures
reproducibility and excellent inhibition over a
wide range of protease classes
• Flexible: Full disclosure of formulations and a
comprehensive selection of specific cocktails
designed to inhibit proteolytic activity from
most tissue or cell type extracts ensures greater
experimental flexibility
3
Application-Specific Protease Inhibitor Cocktails
EMD Millipore inhibitor cocktails are available in a variety of formulations and formats to ensure the best results
on a wide range of starting materials and applications. For example, inhibitor cocktails are available without EDTA
for purification schemes involving metal ion-chelating chromatography or analysis using two dimensional (2D) gel
electrophoresis. Select formulations use recombinant aprotinin for applications that require the use of animal-free
reagents. The table below summarizes the components and application specificity of each protease inhibitor cocktail.
4
Product
Components
Application
Cat. No.
Protease Inhibitor
Cocktail Set I
AEBSF, HCl; Aprotinin, Bovine Lung;
E-64 Protease Inhibitor; EDTA,
Disodium, Leupeptin, Hemisulfate
General Use
80053-844
Protease Inhibitor
Cocktail Set I,
Animal-Free
AEBSF, HCl; Aprotinin, Recombinant;
E-64 Protease Inhibitor; EDTA,
Disodium; Leupeptin Hemisulfate
General Use and for applications which
require the use of animal-free reagents.
80017-342
Protease Inhibitor
Cocktail Set II
AEBSF, HCl; Bestatin; E-64 Protease
Inhibitor; EDTA, Disodium; Pepstatin A
Bacterial cell extracts (except those intended
for metal chelation chromatography)
80053-850
Protease Inhibitor
Cocktail Set III,
EDTA-Free
AEBSF, HCl; Aprotinin, Bovine Lung;
Bestatin; E-64 Protease Inhibitor;
Leupeptin, Hemisulfate; Pepstatin A
Mammalian cells and tissue extracts purified
using metal chelation chromatography;
samples to be analyzed by 2-D gel
electrophoresis
80053-854
Protease Inhibitor
Cocktail Set III,
Animal-Free
AEBSF, HCl; Aprotinin, Recombinant;
Bestatin; E-64 Protease Inhibitor;
Leupeptin, Hemisulfate; Pepstatin A
Mammalian cells and tissue extracts and for
applications which require the use of animalfree reagents
80017-246
Protease Inhibitor
Cocktail Set IV
AEBSF, HCl; E-64 Protease Inhibitor;
Pepstatin A; o-Phenanthroline
Fungal and yeast cell extracts
80503-258
Protease Inhibitor
Cocktail Set V,
EDTA Free
AEBSF, HCl; Aprotinin , Bovine Lung;
E-64 Protease Inhibitor; Leupeptin ,
Hemisulfate
Mammalian cells and tissue extracts purified
using metal chelation chromatography;
samples to be analyzed by 2-D gel
electrophoresis
80601-615
Protease Inhibitor
Cocktail Set V,
Animal-Free
AEBSF, HCl; Aprotinin , Recombinant;
E-64 Protease Inhibitor; Leupeptin ,
Hemisulfate
Mammalian cells and tissue extracts and for
applications which require the use of animalfree reagents
80017-350
Protease Inhibitor
Cocktail Set VI
AEBSF, HCl; Bestatin; E-64 Protease
Inhibitor; Leupeptin, Hemisulfate;
o-Phenanthroline; Pepstatin A
Plant cell extracts
80602-622
Protease Inhibitor
Cocktail Set VII
AEBSF, HCl; Bestatin; E-64
Protease Inhibitor; Pepstatin A;
Phosphoramidon, Disodium Salt
Proteins containing His tag sequences
80602-626
Protease Inhibitor
Cocktail Set VIII
ALLN; Cathepsin Inhibitor I;
E-64 Protease Inhibitor
Broad range cysteine protease inhibition
80603-796
Serine Protease
Inhibitor Cocktail
Set I
AEBSF, HCl; Aprotinin, Bovine Lung;
Elastatinal, GGACK
Broad range serine protease inhibition
80600-148
Protease Inhibitor Set
AEBSF, HCl; E-64; EST; Leupeptin,
Hemisulfate ; Pepstatin A; TLCK,
HCl; TPCK
For screening for suitable protease inhibitors
80053-842
Featured Protease Inhibitor Cocktail
Protease Inhibitor Cocktail Set III, EDTA-Free
Cat. No. 80053-854
Form: Liquid
This cocktail is recommended for use with mammalian cells and tissue extracts and for bacterial cell extracts being used
for metal ion chelation chromatography. Cocktail contains six protease inhibitors (in 1 mL DMSO) with broad specificity
for the inhibition of aspartic, cysteine, and serine proteases as well as aminopeptidases. One mL is sufficient for about
20 g tissue. Note: Available as 1 x 1 mL or as a set of 5 x 1 mL.
Protease Inhibitor
Molecular Weight (MW)
Concentration in Vial
Target Proteases
239.5
100 mM
Serine proteases
Aprotinin, bovine lung
6512
80 µM
Serine proteases and esterases
Bestatin
308.4
5 mM
Aminopeptidease B and leucine aminopeptidase
E-64 Protease Inhibitor
357.4
1.5 mM
Leupeptin, hemisulfate
475.6
2 mM
Cysteine proteases and trypsin-like proteases
Pepstatin A
685.9
1 mM
Aspartic proteases
AEBSF, hydrochloride
Cysteine proteases
More efficient than competitor product:
Volume of cell extract
500 mL
Required amount of EMD Millipore Protease
Inhibitor Cocktail Set III
Required amount of Supplier R Protease
Inhibitor Cocktail
1 mL
10 tablets
5
Protease Inhibitors | Frequently Asked Questions
What criteria should I use when selecting a protease inhibitor?
When processing cells or tissues, you should assume that active proteases are present. Therefore, you should include
protease inhibitors in the early steps of sample preparation. For best results, add protease inhibitors to the protein solution
sample just before use to ensure the maximum activity for the cocktail during sample preparation. Typically, inhibitors
should not be dissolved in buffers and stored for long periods of time. Since different cells and tissue types express
different protease profiles, our application-specific cocktails are formulated with different sets of inhibitors. For example,
serine proteases are widely distributed in all cells; bacterial cells contain higher levels of serine and metalloproteases;
animal tissue extracts are rich in serine, cysteine, and metalloproteases, and plant extracts contain higher quantities of
serine and cysteine proteases. Other selection criteria include what type of method is being used. EDTA is typically not
desirable in 2-D gel electrophoresis studies.
I currently use protease inhibitor cocktail tablets. Is it easy to switch to ready-to-use
liquid cocktails?
Yes, in fact, there are several benefits to using pre-made, liquid, protease inhibitor cocktails. In general, liquid cocktails are:
• S table — our cocktails offer a greater degree of stability compared to tablets dissolved in medium (see Figure 1)
• E fficient — the liquid cocktail format allows immediate exposure of inhibitors to proteases present in samples
• F lexible, minimizing waste — you only need to add the amount of cocktail needed for your specific protocol
•C
onvenient, easy to use — working reagents are dissolved and ready to use
•C
onsistent — the same quantity of inhibitor is always dispensed
I work in an automation lab and want to use the cocktails on a liquid handling workstation.
Are Calbiochem® protease inhibitor cocktails stable?
Our cocktails are compatible for use on liquid handlers and are stable, as shown in the data below.
Calbiochem® Protease Inhibitors offer greater efficiency and stability
Proteolytic Activity
70000
day 0
A = Competitor, 8 °C
60000
day 1
B = Calbiochem® Cocktail VII, 8 °C
50000
day 3
C = Competitor, room temp.
40000
day 5
D = Calbiochem® Cocktail VII, room temp
30000
20000
10000
0
6
No Inhibitors
A
B
C
D
Figure 1. Stability of Protease Inhibitor Dilutions in BugBuster® Lysis Reagent
Protease inhibitors were diluted to the prescribed working concentration (Competitor or Calbiochem® Cocktail VII,
Cat. No. 80602-626). The ability of the inhibitors to inhibit the proteolytic activity of PRONASE® (Cat. No. 80602-612) was measured
by using the Universal HT Protease Assay on days zero, one (24 h post dilution), three (72 h) and five (120 h). The Universal HT
Protease Assay quantifies protease activity using a fluorescein thiocarbamoyl-casein derivative (FTCcasein). Proteolytic activity
liberates FTC-labeled peptides, which results in enhanced fluorescence (Ex.max: 495 nm; Em.max: 525 nm). Addition of the protease
inhibitor cocktails inhibits the proteolytic activity of the PRONASE® (Cat. No. 80602-612), resulting in reduced fluorescence. On day
1, for samples incubated at 8 °C, the competitor tablet inhibited the proteolytic activity by 50% and the Calbiochem® Cocktail VII
inhibited the proteolytic activity by 70%. On day 5, for samples incubated at 8 °C, the competitor tablet caused a 29% decrease in
proteolytic activity in comparison to the Calbiochem® cocktail VII, which caused a 57% decrease in proteolytic activity. The data
show that the efficiency of the cocktail remained higher than the competitor’s tablet in this study.
Key Products
Description
Cat. No.
Protease Inhibitor Cocktail Set I
80053-844
Protease Inhibitor Cocktail Set I, Animal-Free
80017-342
Protease Inhibitor Cocktail Set II
80053-850
Protease Inhibitor Cocktail Set III, EDTA-Free
80053-854
Protease Inhibitor Cocktail Set III, Animal-Free
80017-246
Protease Inhibitor Cocktail Set IV
80503-258
Protease Inhibitor Cocktail Set V, EDTA-Free
80601-615
Protease Inhibitor Cocktail Set V, Animal-Free
80017-350
Protease Inhibitor Cocktail Set VI
80602-622
Protease Inhibitor Cocktail Set VII
80602-626
Protease Inhibitor Cocktail Set VIII
80603-796
Serine Protease Inhibitor Cocktail Set I
80600-148
Protease Inhibitor Set
80053-842
Protease Arrest™ Reagent
80510-638
7
Phosphatase
Inhibitor Cocktails
Phosphorylation and dephosphorylation of proteins mediate signal transduction events that control a
multitude of cellular processes such as cell division, cell proliferation, and apoptosis. Target proteins are
phosphorylated by protein kinases that transfer a phosphate group to a specific protein, typically at serine,
threonine, or tyrosine residues. The covalent attachment of a phosphoryl group changes the conformation
of the protein and alters its ability to interact with a ligand. The phosphate group is removed by the action
of protein phosphatases, which restore the protein to its original dephosphorylated state. For accurate
characterization of the structure and function of phosphorylated proteins, it is critical to preserve their
phosphorylation state during extraction from cell and tissue lysates. Use our phosphatase inhibitor
cocktails and PhosphoSafe™ Extraction Reagent to protect phosphoproteins from different families of
phosphatases.
Application-Specific Phosphatase Inhibitor Cocktails
EMD Millipore inhibitor cocktails are available in a variety of formulations and formats to ensure the best results on a
wide range of starting materials and applications. The table below summarizes the components and application specificity
of each phosphatase inhibitor cocktail.
Product
Components
Application
Phosphatase Inhibitor
Cocktail Set I
(-)-p-Bromotetramisole oxalate;
Cantharidin; Microcystin LR,
Microcystis aeruginosa
Protection against alkaline phosphatases and
Ser/Thr phosphatases such as PP1 and PP2A.
Note: Not available for sale outside of the
United States
80501-128
Phosphatase Inhibitor
Cocktail Set II
Imidazole; Sodium Fluoride; Sodium
Molybdate; Sodium Orthovanadate;
Sodium Tartrate, dihydrate
Protection against acid and alkaline
phosphatases and protein tyrosine
phosphatases (PTPs)
80501-130
Phosphatase Inhibitor
Cocktail Set III
Sodium Fluoride; Sodium
Orthovanadate; Sodium
Pyrophosphate, decahydrate;
b-Glycerophosphate
Protection against Ser/Thr phosphatases and
protein tyrosine phosphatases (PTPs)
80017-332
Phosphatase Inhibitor
Cocktail Set IV
(-)-p-Bromotetramisole oxalate;
Cantharidin; Calyculin A,
Discodermia calyx
Protection against alkaline phosphatases and
Ser/Thr phosphatases such as PP1 and PP2A
101388-172
Protection against Ser/Thr phosphatases and
Protein Tyrosine Phosphatases (PTPs)
82601-054
PhosphoSafe™
Extraction Reagent
8
Cat. No.
Featured Phosphatase Inhibitor Cocktail
Phosphatase Inhibitor Cocktail Set II
Cat. No. 80501-130
Form: Liquid
A cocktail of five phosphatase inhibitors for the inhibition of acid and alkaline phosphatases as well as protein tyrosine
phosphatases (PTPs). Suitable for use with tissue and cell extracts, including extracts containing detergents. Each vial
contains 1 mL aqueous solution of the phosphatase inhibitor cocktail. The concentrations of the individual inhibitors are
shown in the table. Note: 1 set = 5 x 1 mL.
Phosphatase Inhibitor
Molecular Weight (MW)
Concentration in Vial
Target Proteases
Imidazole
68.1
200 mM
Alkaline phosphatase
Sodium fluoride
42.0
100 mM
Acid phosphatase
Sodium molybdate
205.9
115 mM
Acid phosphatase
Sodium orthovanadate
183.9
100 mM
Protein tyrosine phosphatase, alkaline phosphatase
Sodium tartrate, dihydrate
230.1
400 mM
Acid phosphatase
More efficient than competitor product:
Volume of cell extract
500 mL
Required amount of EMD Millipore
Phosphatase Inhibitor Cocktail Set III
Required amount of Supplier R
Phosphatase Inhibitor Cocktail
1 set (5 x 1 mL)
50 tablets
Key Products
Description
Cat. No.
Phosphatase Inhibitor Cocktail Set I
80501-128
Phosphatase Inhibitor Cocktail Set II
80501-130
Phosphatase Inhibitor Cocktail Set III
80017-332
Phosphatase Inhibitor Cocktail Set IV
101388-172
Phosphatase Inhibitor Cocktail Set V
82603-842
PhosphoSafe™ Extraction Reagent
82603-842
9
Related Products
Description
80031-734
BugBuster® Master Mix Extraction reagent
82601-026
BugBuster® Plus Benzonase Nuclease
80108-830
BugBuster® Plus Lysonase™ Kit
80602-814
BugBuster® HT Protein Extraction Reagent
80500-206
BugBuster® 10X Protein Extraction Reagent
80500-202
BugBuster®(primary amine-free) Extraction Reagent
80508-842
PopCulture® Reagent
80600-006
Insect PopCulture® Reagent
80601-830
YeastBuster™ Protein Extraction Reagent
60801-624
CytoBuster™ Protein Extraction Reagent
80509-652
NucBuster™ Protein extraction Reagent
80601-632
ProteoExtract® Subcellular Protein Extraction Kit
80601-632
ProteoExtract® Native Membrane Protein Extraction Kit
80016-460
ProteoExtract® Transmembrane Protein Extraction Kit
80017-516
ProteoExtract® Complete Mammalian Protein Extraction Kit
80601-840
ProteoExtract® Cytosol/Mitochondria Fractionation Kit
ProteoExtract® Tissue Dissociation Kit
10
Cat. No.
BugBuster® Protein Extraction Reagent
80511-866
200066-520
ProteoExtract® All-in-One Trypsin Digestion Kit
80603-586
ProteoExtract® Phosphopeptide Enrichment TiO2 Kit
82603-358
ProteoExtract® Phosphopeptide Enrichment SCIMAC Kit
82603-360
11
www.emdmillipore.com
BugBuster, PopCulture, ProteoExtract, Calbiochem, Amicon, and PRONASE are registered trademarks of
Merck KGaA, Darmstadt, Germany.
EMD Millipore, the M mark, YeastBuster, CytoBuster, NucBuster, PhosphoSafe, PureProteome,
and Protease Arrest™ are trademarks of of Merck KGaA, Darmstadt, Germany.
Lit No. PB4994EN00 LS-SBU-11-05727 Printed in the USA 02/2012
© 2012 EMD Millipore Corporation, Billerica, MA, USA. All rights reserved.
0212 3M Lit. No. 05105