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Protease Inhibitor Cocktail (EDTA free)

The inhibition of intra and extra cellular proteases is vital to purify and collect the expressed proteins. Saving trouble of finding adequate inhibitors, a wide range of proteases are inhibited by the Protease Inhibitor Cocktail. This product does not interfere with metalloproteins since it is EDTA free.

  • Wide range: Contains inhibitors for a variety of serin and cysteine proteases
  • Contains no EDTA: Efficient for purification of metalloproteins and His tag gene exchanged proteins
  • Easy to use: Dilute according to your need (100X stock concentration)



Application 1
Comparison of EDTA containing and EDTA free inhibitor activity for Thermolysin
Protease activity had been measured by Hagiwara method(1) and Burnett method(2).
  1. Dissolve adequate quantity of the enzyme in 50mM Tris Chloride Salt solution (pH7.6) and create dilution series in 100 µl each.
  2. Add 400µl of substrate (1.25% of Asocasein in 50mM Tris Chloride Salt solution (pH 7.6) ) to each of the 100 µl of diluted solutions, mix and stir.
  3. Incubate at 37°C for time suitable for the enzyme.
  4. Add 1ml of stop solution (10% Trichloroacetic Acid) and vortex vigorously.
  5. Centrifuge 19000 x g, for 5 minutes and collect supernatant.
  6. Measure absorption of the supernatant at 335 nm. (Higher absorption indicates the presence of free
    Tyrosine, product of not inhibited substrate degradation.)
Thermolysin (from Bacillus thermoproteolyticus)
is a metalloprotease. The compared protease inhibitors do not inhibit Thermolysin activity. The presence of EDTA in the common product inhibits Thermolysin, while the EDTA free product does not cause damage.


The decrase in Thermolysin activity (in %) in the presence of protease inhibitor cocktails.
Product name Thermolysin (µg/ml)
0.8 1.6 6.4 12.8
Protease Inhibitor Cocktail for General Use 100.0% 99.6% 99.2% 99.7%
Protease Inhibitor Cocktail (EDTA free) 9.7% 8.2% 8.0% 6.3%



Application 2

Enzyme inhibition
Inhibition efficiency of Protease Inhibitor Cocktail (EDTA free)

Origin of Protease Protease Protease Inhibitor Cocktail (EDTA free) (Protease concentration)
Papain (from papaya) Cystein 100% (2.5 µg/ml)
 96.1% (5.0 µg/ml) 96.5% (20 µg/ml)
Trypsin (from porcine pancreas) Serine 96.9% (2.0 µg/ml) 94.0% (4.0 µg/ml) 88.1% (8.0 µg/ml)
Chymotrypsin (from bovine pancreas) Serine 95.1% (0.75 µg/ml) 90.5% (1.5 µg/ml) 83.1% (3.0 µg/ml)
Pronase (from Streptomyces griseus) Mixture 96.5% (0.6 µg/ml) 95.3% (1.2 µg/ml) 92.3% (2.4 µg/ml)
Pancreas extract (bovine) Mixture 89.8% (5.0 µg/ml) 87.2% (10 µg/ml) 85.6% (20 µg/ml)



Composition

The product (100 times concentrated stock solution) contains:
Inhibitors Targeted protease
50 µmol / l 4-(2-Aminoethyl)benzenesulfonyl Fluoride Hydrochloride (AEBSF) Serine protease
15 µmol / l Aprotinin Serine protease, elastase
100 µmol / l E-64 Cystein protease
100 µmol / l Leupeptin Hemisulfate Monohydrate Cystein protease, trypsin like protease



Protocol
  1. Dissolve the powdered product by adding 1 ml de-ionized water into the bottle to make the 100 times concentrated stock solution.

  2. Dilute it with the your choice of buffer to make 100 ml.



References
  1. B. Hagiwara, H. Matsubara, M. Nagaki and K. Okunuki, Crystalline bacterial protease I.
    Preparation of crystalline proteinase of Bacillus subtilis. J. Biochem.,45,185-194(1958)

  2. T. J. Burnett, G. W. Shankweiler and J. H. Hageman, Activation of intracellular serine
    proteinase in Bacillus subtilis cells during sporulation. J. Bacteriol, 165,139-145, (1986)



Caution
  • In case of very high protease concentration, use higher concentration of the product (dilute only up to between 50 and 20 times).
  • Keep the inhibitor cocktail stock solution below -20°C, and use it within 3 months.



Expiration

For powder in unopened bottle: two years from manufacturing. Expiration date is stated on the
product label (Exp. yy / mm)



Ordering information

Product name
 Grade Storage Code No.
 Quantity
Protease Inhibitor Cocktail (EDTA free) (100X)
 SP F 03969-21 5 x 1ml
(Storage)
A = Cool and dark, RT = Room temperature, F = Freezer, R = Refrigerator


Printed version(PDF 108KB)

(Related productd)
Product name
 Grade Storage Code No.
 Quantity
Protease Inhibitor Cocktail for general use (100X)
 SP
 F 04080-11 5 x 1ml
Protease Inhibitor Cocktail
for Use with Mammalian Cell Extracts
 SP
 F 25955-11 5 x 1ml
For research use only, not intended for diagnostic or drug use.
(Storage)
A = Cool and dark, RT = Room temperature, F = Freezer, R = Refrigerator



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