Kelli Whiteman
last modified: 8/6/02
Proteinase K facts:
Cleaves after hydrophobic amino acid residues
Recommended working concentration: 100 ug/ml = 0.0001 g/ml (Sigma)
Active at pH 4.3-12.0, optimal activity at pH 8.0
Active between 20-60ºC
Active in [GdnHCl] or [urea] below 2 M
Active in SDS to 0.5%
MW = 28,900 Da
Methods:
Start boiling water on hot plate. Prepare tubes with 10 ul of 2% SDS loading
buffer. Label tubes for each sample time point. Place floating foam card and
tube toppers nearby.
Each reaction sample should contain 2 ug of protein, the same amount of protease, and enough buffer for a total sample volume of 15 uL. The buffer is 20 mM Tris pH 8.0 with 100 mM NaCl.
Incubate samples for varying times, stopping at 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 90, 105, 120 minutes. (May need to consider adding additional time points out to 3 hrs, along with points at 2.5, 7.5, 12.5 min.)
Inhibit enzyme activity by adding 15 uL-sized samples from reaction tube to 10 uL of 2% SDS loading buffer and boiling for 10 minutes.
Analyze by 14% SDS-PAGE using
silver staining. Also run the following standards:
Native Pertactin (1-15)
Proteinase K (15-10)
MW Markers (1-15)
Truncated P.69 (1-15)space.