Methods in Molecular Biology • 16 Enzymes of Molecular Biology

Proteinase K 309

However, misleading results can be obtained if exposed regions of the
polypeptide are inherently protease resistant. To differentiate genuine
protection from protease activity and inherent protease activity, a fur-
ther digestion is carried out in the presence of a nonionic detergent
(e.g., 0.5-2% Triton X- 100), which disrupts membranes allowing access
of all proteins to proteinase K. Any polypeptide or fragment remaining
after this treatment must be inherently protease resistant. The ability
of proteinase K to function in the presence of detergents, and its broad
specificity, makes the enzyme a very appropriate one for this type of
study. Digestion of membrane is carried out at a range of concentra-
tions (generally 20-1000 lag/mL) to find optimum conditions. Pro-
teinase K is normally prepared fresh as a 10X stock solution in 100 mM
CaC12. Following digestion, it is necessary to inhibit the enzyme to
prevent further degradation of proteins following protein extraction
for SDS gel electrophoresis.

References

1. Ebeling, W., Hennrich, N., Klockow, M., Metz, H., Orth, H. D., and Lang, H.
   (1974) Proteinase K from Tritirachium album Limber. Eur. J. Biochem. 47,
   91-97.


2. Mathew, C. G. P. (1984) Isolation of high molecular weight eukaryotic DNA,
   in Methods in Molecular Biology, vol. 2: Nucleic Acids (Walker, J. M., ed.),
   Humana, Clifton, NJ, pp. 31-34.


3. Steven, J., McKechnie, D., and Graham, A. (1988) Isolation of high molecular
   weight DNA suitable for the construction of genomic libraries, in Methods in
   Molecular Biology, vol. 4: New Nucleic Acid Techniques (Walker, J. M., ed.),
   Humana, Clifton, NJ, pp. 221-234.


4. Haley, J. D. (1988) Cosmid library construction, in Methods in Molecular Biol-
   ogy, vol. 4: New Nucleic Acid Techniques (Walker, J. M. ed.), Humana, Clifton,
   NJ, pp. 257-283.


5. Van Helden, P. D. and Hoal, E. G. (1988) Plasmid preparation on Sephacryl
   S 1000, in Methods in Molecular Biology, vol. 4: New Nucleic Acid Techniques
   (Walker, J. M., ed.), Humana, Clifton, NJ, pp. 69-74.


6. B ateson, A. N. and Pollard, J. W. (1988) Construction of mammalian genomic
   libraries using ~, replacement vectors, in Methods in Molecular Biology, vol.
   4: New Nucleic Acid Techniques (Walker, J. M.ed.), Humana, Clifton, NJ, pp.
   235-355.


7. Plumb, M. A. and Goodwin, G. H. (1988) Detection of sequence-specific pro-
   tein-DNA interaction by the DNA-footprinting techniques, in Methods in Mo-
   lecularBiology, vol. 4: New Nucleic Acid Techniques (Walker, J. M., ed.), Humana,
   Clifton, NJ, pp. 139-164.