protease inhibitors - biovision...protease inhibitors and cocktails which can prevent in vitro...
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PROTEASE Inhibitors
www.biovision.com | 800-891-9699
Proteases 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 PropertiesProduct Cat. No. Sizes Target Protease Solubility/Suggested working concentration Comments
AEBSF, hydrochloride 1644-200, 1644-1000
200 mg1 g
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 4690-54690-1004690-1000
5 mg 100 mg1 g
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 1733-251733-100
25 mg100 mg
Aminopeptidases • Soluble in MeOH • Working concentration: 1-10 µM
Does not inhibit carboxypeptidases
E-64 1739-51739-25
5 mg25 mg
Cysteine proteases • Soluble in H2O • Working concentration: 1-10 µM
-
Leupeptin, hemisulfate 1648-251648-501648-100
25 mg50 mg100 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 1760-101760-50
10 mg50 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 1732-251732-100
25 mg100 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 CocktailsProduct 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 ml5 ml 1 set (5 x 1 ml)
AEBSF hydrochloride, Aprotinin, bovine lung, Bestatin, E-64, 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 1 set ( 1x 5 ml) AEBSF hydrochloride, Aprotinin, bovine lung, E-64, EDTA, Disodium, and Leupeptin hemisulfate
Cysteine, Serine, Trypsin-like proteases, Aminopeptidases, Metalloproteases, and Esterases
A general use cocktail. Reconstitution in DMSO provides a 100X stock solution
EZBlock™ Protease Inhibitor Cocktail III K278- 1EA 1 set ( 1x 5 ml) AEBSF hydrochloride, Bestatin, E-64, EDTA, Disodium, and Pepstatin A
Aspartic, Cysteine, Serine proteases, Aminopeptidases, and Metalloproteases
For use in bacterial cell extracts (where no metal ion chromatography is used). The cocktail should be reconstituted in DMSO: water mixture
EZBlock™ Protease Inhibitor Cocktail IV K279- 1K279-1EA
1 ml, 1 set ( 5 x 1 ml)
AEBSF hydrochloride, E-64, Pepstatin A, and o-Phenanthroline
Aspartic, Cysteine, Serine proteases, and Metalloproteases
For use in fungal and yeast cell extracts
PHOSPHATASE Inhibitors
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Protein 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 InhibitorsProduct 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 CocktailsProduct Cat. No. Sizes Inhibitors Target Phosphatases Comments
EZBlock™ Phosphatase Inhibitor Cocktail I K273-1K273-1EA
1 ml1 set (5 x 1 ml)
Sodium fluoride, Sodium orthovanadate, Sodium pyrophosphate decahydrate, and b-Glycerophosphate
Acid phosphatases, Alkaline phosphatases, Ser/Thr phosphatases, and Protein Tyrosine Phosphatases
Provided as a 50X stock solution in H2O
EZBlock™ Phosphatase Inhibitor Cocktail II K275-1K275-1EA
1 ml1 set (5 x 1 ml)
Imidazole, Sodium fluoride, Sodium molybdate, Sodium orthovanadate, and Sodium tartrate
Acid phosphatases, Alkaline phosphatases, Ser/Thr phosphatases, and Protein Tyrosine Phosphatases
Provided as a 100X stock solution in H2O
EZBlock™ Phosphatase Inhibitor Cocktail III K276-1K276-1EA
1 ml1 set (5 x 1 ml)
p-Bromotetramisole oxalate, Cantharidin, and Calyculin A
Alkaline phosphatases and Ser/Thr phosphatases (PP1 and PP2A)
Provided as a 50X stock solution in DMSO
EZBlock™ Phosphatase Inhibitor Cocktail IV K282-1K282-1EA
1 ml1 set (5 x 1 ml)
Imidazole, b-Glycerophosphate, Sodium fluoride, Sodium molybdate, Sodium orthovanadate, and Sodium tartrate
Acid phosphatases, Alkaline phosphatases, Ser/Thr phosphatases, and Protein Tyrosine Phosphatases
Provided as a 100X stock solution in H2O
EZBlock™ Universal Protease and Phosphatase Inhibitor CocktailsProduct Cat. No. Sizes Inhibitors Target Proteases/Phosphatases Application/Comments
EZBlock™ Universal Protease and Phosphatase Inhibitor Cocktail
K283-1K283-1EA
1 ml1 set (5 x 1 ml)
Aprotinin, Bestatin, E-64, Leupeptin, 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.
EZBlock™ Universal Protease and Phosphatase Inhibitor Cocktail, EDTA-Free
K284-1K284-1EA
1 ml, 1 set (5 x 1 ml)
Aprotinin, Bestatin, E-64, Leupeptin 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.
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).
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