M.B. Enzymes GmbH

Proteinase K.

Description

Proteinase K (EC 3.4.21.14) is isolated from fungus Tritirachium album is a highly active serine protease of the subtilisin type. The enzyme catalyzes hydrolysis of a wide variety of peptide bonds but exhibits a preference for peptide bonds C-terminal to aromatic and uncharged amino acids. M.W. approx. 28930 Da. Activity >30U/mg protein.

Unit definition

One Anson unit liberates 1µM of Folin-positive amino acids per min at pH 7.5 and 35°C using haemoglobin as substrate.

Reaction buffers

#B29 (x10): 500 mM Tris-HCl pH 7.5, 50 mM CaCl₂. #B30 (x10, for isolation of DNA and RNA isolation from blood, cells and tissue): 100 mM Tris-HCl pH 8.0, 50 mM EDTA, 5% SDS.Use enzyme in excess (30-45U) to degrade 100 mg of solid tissue, pellet from 20 ml blood or 5x10⁷ mammalian cells (1 ml suspension). The working concentration of the enzyme should be 3-6U/ml in 10-15 ml of the lysis buffer. Incubation time can range from 30 min to 18 h depending on the nature of the experiment.

Reaction conditions

Proteinase K is stable over a wide pH range (4.0-12.5); however, the enzyme is normally used in pH range 7.5-9.0. The activity of Proteinase K is severalfold higher at 50°C than at 37°C.The activity can be stimulated addition of denaturing agents (0.5% SDS, 1% Triton X-100 and 1-4 M urea). The enzyme is inactivated by Pefabloc SC and inhibited by diisopropyl phosphofluoridate (DFP) and phenylmethanesulfonyl fluoride (PMSF).

When Ca²⁺ is removed from the enzyme, some of the catalytic activity is lost because of long-range structural chanages. Because EDTA is a component of the buffer solutions used for RNA/DNA isolation, digestion is usually carried out under non optimal conditions. However, residual activity in a presence of EDTA is sufficient to degrade most proteins.

Storage

Store at -20°C.

Applications

preparation of high-molecular weight DNA for PFGE and intact cellular RNA; protein fingerprinting; removal of nucleases from preparations of DNA and RNA; to study membrane protein topology and protein translocation across membranes.

Quality control

activity, SDS-PAG purity, no contaminating nuclease activity.

References

1. Ebeling, W. (1974) Eur. J. Biochem. 47, 91-97. 2. Bajorath, J. et al. (1988) Eur. J. Biochem. 176, 441-447.

Catalog #

P3.3

P3.5

Pack size

500 U

2500 U

Proteinase K.
Description	Proteinase K (EC 3.4.21.14) is isolated from fungus Tritirachium album is a highly active serine protease of the subtilisin type. The enzyme catalyzes hydrolysis of a wide variety of peptide bonds but exhibits a preference for peptide bonds C-terminal to aromatic and uncharged amino acids. M.W. approx. 28930 Da. Activity >30U/mg protein.
Unit definition	One Anson unit liberates 1µM of Folin-positive amino acids per min at pH 7.5 and 35°C using haemoglobin as substrate.
Reaction buffers	#B29 (x10): 500 mM Tris-HCl pH 7.5, 50 mM CaCl₂. #B30 (x10, for isolation of DNA and RNA isolation from blood, cells and tissue): 100 mM Tris-HCl pH 8.0, 50 mM EDTA, 5% SDS.Use enzyme in excess (30-45U) to degrade 100 mg of solid tissue, pellet from 20 ml blood or 5x10⁷ mammalian cells (1 ml suspension). The working concentration of the enzyme should be 3-6U/ml in 10-15 ml of the lysis buffer. Incubation time can range from 30 min to 18 h depending on the nature of the experiment.
Reaction conditions	Proteinase K is stable over a wide pH range (4.0-12.5); however, the enzyme is normally used in pH range 7.5-9.0. The activity of Proteinase K is severalfold higher at 50°C than at 37°C.The activity can be stimulated addition of denaturing agents (0.5% SDS, 1% Triton X-100 and 1-4 M urea). The enzyme is inactivated by Pefabloc SC and inhibited by diisopropyl phosphofluoridate (DFP) and phenylmethanesulfonyl fluoride (PMSF). When Ca²⁺ is removed from the enzyme, some of the catalytic activity is lost because of long-range structural chanages. Because EDTA is a component of the buffer solutions used for RNA/DNA isolation, digestion is usually carried out under non optimal conditions. However, residual activity in a presence of EDTA is sufficient to degrade most proteins.
Storage	Store at -20°C.
Applications	preparation of high-molecular weight DNA for PFGE and intact cellular RNA; protein fingerprinting; removal of nucleases from preparations of DNA and RNA; to study membrane protein topology and protein translocation across membranes.
Quality control	activity, SDS-PAG purity, no contaminating nuclease activity.
References	1. Ebeling, W. (1974) Eur. J. Biochem. 47, 91-97. 2. Bajorath, J. et al. (1988) Eur. J. Biochem. 176, 441-447.
Catalog #	P3.3	P3.5
Pack size	500 U	2500 U