Download Protease and Phosphatase Inhibitor Cocktails

PROTEASE Inhibitors
P
roteases are a family of naturally occurring regulatory enzymes that
cleave the peptide bonds between the amino acids of polypeptide
chains. They can be classified into six broad groups depending on the
amino acid at which they cleave. These include Serine proteases, Threonine
proteases, Cysteine proteases, Aspartate proteases, Metalloproteases, and
Glutamic acid proteases. Based on the optimal pH of their activity, they are
sub-classified into acid proteases, neutral proteases and alkaline proteases.
Protease inhibitors are compounds that inhibit specific proteases (mentioned
above). A delicate equilibrium of these proteases and their respective
inhibitors can control proteolytic cascades, thereby regulating key events
like coagulation, inflammation and acute phase response in the body.
During protein expression and isolation, endogenous cellular proteases can
rapidly digest cellular proteins resulting in substantially diminished quality
and quantity of protein samples needed for characterization and analysis.
BioVision has introduced a new and exciting line of general, as well as specific
protease inhibitors and cocktails which can prevent in vitro proteolysis events.
These protease inhibitors and cocktails are provided in an easy-to-use form
and can be employed for inhibition of a broad range of proteases. The inhibitor
cocktail kits are designed to include various combinations of inhibitors at their
optimum concentrations, providing researchers with a number of options to
specifically choose from, including the EDTA-free form.
Some commonly used Protease Inhibitors and their Properties
Product
Cat. No.
Sizes
Target Protease
Solubility/Suggested working concentration
Comments
AEBSF, hydrochloride
1644-200,
1644-1000
200 mg
1g
Serine proteases
• Soluble in H2O
• Working concentration: 0.1 -5 mM in cell culture; in
other applications: 0.1 -0.25 µM
Aqueous solutions are stable for several
months between pH 5.0 – 6.0; limited
stability above pH 7.5
Aprotinin, bovine lung
4690-5
4690-100
4690-1000
5 mg
100 mg
1g
Serine proteases and
esterases
• Soluble in H2O, aqueous buffers
• Working concentration: In cell culture: 0.01-3 µg/
ml; in other applications: 10-250 µg/ml
Sterile filtered solutions at pH 5.0-8.0 are
stable for several months. Denatures at
pH>12. Avoid repeated freeze thawing
Bestatin
1733-25
1733-100
25 mg
100 mg
Aminopeptidases
• Soluble in MeOH
• Working concentration: 1-10 µM
Does not inhibit carboxypeptidases
E-64
1739-5
1739-25
5 mg
25 mg
Cysteine proteases
• Soluble in H2O
• Working concentration: 1-10 µM
-
Leupeptin, hemisulfate 1648-25
1648-50
1648-100
25 mg
50 mg
100 mg
Serine and thiol
proteases
• Soluble in H2O
• Working concentration: 1-100 µM
Stable for several months when stored in
aliquots at -20°C
Nafamostat mesylate
1760-10
1760-50
10 mg
50 mg
Serine proteases
• Soluble in H2O or DMSO
• Working concentration: 50 µg/ml
Stable for 4 weeks if stored at 4°C and
several months if stored at -80°C
Pepstatin A
1732-25
1732-100
25 mg
100 mg
Aspartic proteases
•Soluble in DMSO or MeOH
• Working concentration: 1-10 µM
-
PMSF
1548-5
5 mg
Serine proteases
• Soluble in isopropanol
• Working concentration: 0.1-1 µM
Unstable in aqueous solutions; add fresh
at each step
EZBlock™ Protease Inhibitor Cocktails
Product
Cat. No.
Sizes
Inhibitors
Target Proteases
Application/Comments
Protease Inhibitor Cocktail, EDTA-Free
K271-500
1 kit
Aprotinin, bovine lung, Leupeptin
hemisulfate, Pepstatin A, and
PMSF
Aspartic, Cysteine, Serine
proteases, and esterases
A general use cocktail. Reconstitution
in DMSO provides a 500X stock solution
EZBlock™ Protease Inhibitor Cocktail,
EDTA-Free
K272-1
K272-5
K272-1EA
1 ml
AEBSF hydrochloride, Aprotinin,
5 ml
bovine lung, Bestatin, E-64,
1 set (5 x 1 ml) Leupeptin hemisulfate, and
Pepstatin A
Aspartic , Cysteine, Serine,
Trypsin-like proteases,
Aminopeptidases and Esterases
For use in mammalian cell and
tissue extracts and for bacterial
cell extracts using metal chelation
chromatography. Provided as a 200X
stock solution
EZBlock™ Protease Inhibitor Cocktail II
K277- 1EA
Cysteine, Serine, Trypsin-like
1 set ( 1x 5 ml) AEBSF hydrochloride, Aprotinin,
bovine lung, E-64, EDTA, Disodium, proteases, Aminopeptidases,
and Leupeptin hemisulfate
Metalloproteases, and Esterases
EZBlock™ Protease Inhibitor Cocktail III K278- 1EA
1 set ( 1x 5 ml) AEBSF hydrochloride, Bestatin,
E-64, EDTA, Disodium, and
Pepstatin A
EZBlock™ Protease Inhibitor Cocktail IV K279- 1
K279-1EA
1 ml,
AEBSF hydrochloride, E-64,
Aspartic, Cysteine, Serine
1 set ( 5 x 1 ml) Pepstatin A, and o-Phenanthroline proteases, and Metalloproteases
www.biovision.com | 800-891-9699
A general use cocktail. Reconstitution
in DMSO provides a 100X stock solution
Aspartic, Cysteine, Serine
For use in bacterial cell extracts (where
proteases, Aminopeptidases, and no metal ion chromatography is used).
Metalloproteases
The cocktail should be reconstituted in
DMSO: water mixture
For use in fungal and yeast cell extracts
PHOSPHATASE Inhibitors
P
rotein phosphorylation is the most common and important form of
reversible protein posttranslational modification (PTM), with up to
30% of all proteins being phosphorylated at any given time. Protein
kinases (PKs) are the effectors of phosphorylation and catalyze the transfer
of a γ-phosphate group from ATP to specific amino acids on proteins. Proteins
are phosphorylated predominantly on Ser, Thr and Tyr residues, which
account for 86, 12 and 2% respectively of the phosphoproteome, at least in
mammals. In contrast, protein phosphatases (PPs) are the primary effectors of
dephosphorylation, and are responsible for restoring the protein to its original
dephosphorylated state. Phosphatases can be classified as non-specific
phosphatases (e.g. acid or alkaline phosphatases), serine/threonine-specific,
tyrosine-specific and dual-specific phosphatases (Ser/Thr & Tyr).
A wide range of biological processes are controlled by phosphorylation
and dephosphorylation of proteins. In order to understand the accurate
phosphorylated status of a protein, it is important to preserve the
phosphorylation state of the proteins during the extraction of phosphoproteins
from tissue and cell sample lysates. BioVision introduces four different
EZBlock™ Phosphatase Inhibitor Cocktails and two new Universal Protease
and Phosphatase inhibitor cocktails which would provide protection against
both proteases and phosphatases during protein extraction and purification.
Phosphatase Inhibitors
Product
Cat. No.
Sizes
Target Phosphatase
Solubility
bpV(phen)
1793-5
5 mg
Protein Tyrosine Phosphatase (PTP) and PTEN
DMSO or H2O
bpV(pic)
1794-5
5 mg
Protein Tyrosine Phosphatase (PTP) and PTEN
DMSO or H2O
Calyculin A
1562-025
25 µg
Ser/Thr phosphatases (PP1 and PP2)
DMSO or EtOH
Cyclosporine A
1522-100, 1522-1G
100 mg, 1 g
Cyclosporine-Cyclophilin complex inhibits PP2B (Calcineurin)
DMSO or EtOH
FK-506
1563-1, 1563-10
1 mg, 10 mg
Protein phosphatase 2B
DMSO or EtOH
Okadaic Acid
1543-025
25 µg
Ser/Thr phosphatases (PP1, PP2A and PP2B)
DMSO or EtOH
Okadaic Acid Ammonium Salt
1766-025
25 µg
Ser/Thr phosphatases (PP1, PP2A and PP2B)
DMSO or EtOH
Okadaic Acid Potassium Salt
1765-025
25 µg
Ser/Thr phosphatases (PP1, PP2A and PP2B)
DMSO or EtOH
Okadaic Acid Sodium Salt
1764-025
25 µg
Ser/Thr phosphatases (PP1, PP2A and PP2B)
DMSO or EtOH
VO-OHpic
1801-5, 1801-25
5 mg, 25 mg
PTEN
H2O
EZBlock™ Phosphatase Inhibitor Cocktails
Product
Cat. No.
Sizes
Inhibitors
Target Phosphatases
Comments
EZBlock™ Phosphatase Inhibitor Cocktail I
K273-1
K273-1EA
1 ml
Sodium fluoride, Sodium orthovanadate, Acid phosphatases, Alkaline
Provided as a 50X stock
1 set (5 x 1 ml) Sodium pyrophosphate decahydrate, and phosphatases, Ser/Thr phosphatases, solution in H2O
b-Glycerophosphate
and Protein Tyrosine Phosphatases
EZBlock™ Phosphatase Inhibitor Cocktail II
K275-1
K275-1EA
1 ml
Imidazole, Sodium fluoride, Sodium
1 set (5 x 1 ml) molybdate, Sodium orthovanadate, and
Sodium tartrate
Acid phosphatases, Alkaline
Provided as a 100X
phosphatases, Ser/Thr phosphatases, stock solution in H2O
and Protein Tyrosine Phosphatases
EZBlock™ Phosphatase Inhibitor Cocktail III K276-1
K276-1EA
1 ml
p-Bromotetramisole oxalate, Cantharidin, Alkaline phosphatases and Ser/Thr
1 set (5 x 1 ml) and Calyculin A
phosphatases (PP1 and PP2A)
Provided as a 50X stock
solution in DMSO
EZBlock™ Phosphatase Inhibitor Cocktail IV K282-1
K282-1EA
1 ml
Imidazole, b-Glycerophosphate, Sodium Acid phosphatases, Alkaline
Provided as a 100X
1 set (5 x 1 ml) fluoride, Sodium molybdate, Sodium
phosphatases, Ser/Thr phosphatases, stock solution in H2O
orthovanadate, and Sodium tartrate
and Protein Tyrosine Phosphatases
EZBlock™ Universal Protease and Phosphatase Inhibitor Cocktails
Product
Cat. No.
Sizes
EZBlock™ Universal Protease and
Phosphatase Inhibitor Cocktail
K283-1
K283-1EA
EZBlock™ Universal Protease and
Phosphatase Inhibitor Cocktail,
EDTA-Free
K284-1
K284-1EA
Inhibitors
Target Proteases/Phosphatases
Application/Comments
1 ml
Aprotinin, Bestatin, E-64, Leupeptin,
1 set (5 x 1 ml) b-Glycerophosphate, Sodium
fluoride, Sodium orthovanadate,
Sodium pyrophosphate and EDTA,
Disodium.
Cysteine proteases, Serine proteases,
trypsin-like proteases, esterases,
Aminopeptidases, Metalloproteases,
Acid phosphatases, alkaline
phosphatases, Ser/Thr phosphatases,
and Protein Tyrosine Phosphatases
Provided as a 10X aqueous
solution. This cocktail does not
contain AEBSF and thus should
be mass spectrometry (MS)
compatible.
1 ml,
Aprotinin, Bestatin, E-64, Leupeptin
1 set (5 x 1 ml) hemisulfate, b-Glycerophosphate,
Sodium fluoride, Sodium
orthovanadate, and Sodium
pyrophosphate
Cysteine proteases, Serine proteases,
trypsin-like proteases, esterases,
Aminopeptidases, Acid phosphatases,
alkaline phosphatases, Ser/Thr
phosphatases, and Protein Tyrosine
Phosphatases
Provided as a 10X aqueous
solution. This cocktail does not
contain AEBSF and thus should
be mass spectrometry (MS)
compatible.
www.biovision.com | 800-891-9699
www.biovision.com
USA
BioVision, Inc.
155 South Milpitas Blvd.
Milpitas, CA 95035
Phone: 800-891-9699
Customer Service
[email protected]
Technical Support
[email protected]
To Place an Order
[email protected]
OEM or Bulk Order Inquiries
[email protected]
BioVision’s EZBlock™ Phosphatase Inhibitor Cocktail I (50X), Cat# K273-1 was used to test it’s ability to inhibit purified Alkaline
Phosphatase (Calf thymus) and Acid Phosphatase (wheat germ) using methylumbelliferyl phosphate disodium (MUP) as a
substrate. In each reaction, MUP was used at 500 pmol/well and our EZBlock™ Phosphatase Inhibitor Cocktail I (50X) was added
at 0, 50-fold (50X), 25-fold (25X) or 10-fold (10X) dilution into the final reaction volume. Equal amounts of Alkaline Phosphatase
or Acid Phosphatase were added to their respective wells and the amount of MUP hydrolyzed was detected fluorometrically (Ex/
Em = 360/440 nm). BioVision’s EZBlock™ Phosphatase Inhibitor Cocktail I (50X) used at 50X, 25X and 10X dilutions inhibited
Alkaline Phosphatase activity by 85 %, 90 % and 96 %, respectively and that of Acid Phosphatase activity by 80 %, 85% and 96
%, respectively. Vehicle Control (DMSO) had no effect (data not shown).