New England Biolabs, P8107S, Proteinase K, Molecular Biology Grade

Proteinase K is a subtilisin-related serine protease that hydrolyzes a variety of peptide bonds and is frequently used to cleanup enzymatic reactions or cell lysates. Related Categories Proteases,, Nucleic Acid Purification Specification Unit Definition One unit will digest urea-denatured hemoglobin at 37°C (pH 7.5) per minute to produce equal absorbance as 1.0 μmol of L-tyrosine using Folin & Ciocalteu's phenol reagent (6). Storage Buffer 20 mM Tris-HCl 1 mM CaCl2 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 95°C for 10 minutes Molecular Weight Theoretical: 28.9 kDa Unit Assay Conditions 0.5–2 μg of Proteinase K is incubated with 2% denatured hemoglobin solution for 10 minutes at 37°C (pH 7.5). After precipitation, neutralization and addition of Folin & Ciocalteu's phenol reagent, absorbance of soluble cleavage products are measured at 750 nm. Absorbance is compared to a standard curve of L-tyrosine absorbance prepared similarly. FAQ Q: The protocol I am using requires a µg/ml concentration of Proteinase K. What is the protein concentration of Proteinase K, Molecular Biology Grade? A: The protein concentration of Proteinase K, Molecular Biology Grade is ~20 mg/ml and its unit activity is 800 U/ml. Q: What buffer should I use for Proteinase K? A: Proteinase K is active in a wide range of buffers including all Restriction Enzyme NEBuffers, Q5 Reaction Buffer, OneTaq Standard Reaction Buffer, Standard Taq Reaction Buffer and RNAPol Reaction Buffer. Proteinase K is active with an optimal pH between 7.5 and 12.0 (Ebeling et al., 1974) and temperatures between 20 and 60°C (Bajorath et al., 1988). Activity is stimulated when up to 2% SDS or up to 4 M Urea are included in the reaction (Hilz et al., 1975). Calcium is important for thermostability of Proteinase K but it is not required for catalysis, therefore Proteinase K is also active in buffers containing chelating agents such as EDTA (Bajorath et al., 1988). Q: If I need to dilute Proteinase K, Molecular Biology Grade, how should I do this? A: For immediate use, Proteinase K can be diluted into 1X reaction buffer. To create a stock that permits longer-term storage, the dilution buffer should contain Ca++ (1mM is typically sufficient). Q: Why is the unit definition assay for Thermolabile Proteinase K (NEB #P8111) different from the unit definition assay for Proteinase K, Molecular Biology Grade (NEB # P8107)? A: Proteinase K, Molecular Biology Grade (#P8107) uses a traditional denatured hemoglobin substrate to measure activity (1). This traditional assay allows comparison of Proteinase K activity to the current and historical market. Over the years, casein has also been used as a substrate with less precision then hemoglobin. Thermolabile Proteinase K (#P8111) uses a more modern approach in which a nitroanilide substrate is used to measure activity by visible absorbance. This assay is preferred for its direct readout of proteolysis, sensitivity and 96-well plate compatibility. For the development of the direct assay see Pozgay et al. (2). References Anson, M. L. et al. (1938) J. Gen. Physiol., 22, 78-89. “The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin.” Pozsgay, M. et al. (1979) Eur. J. Biochem., 95, 115-119 “A method for designing peptide substrates for proteases. Tripeptidyl-p-nitroanilide substrates for Subtilisin Carlsberg” Q: What is the activity if Proteinase K, Molecular Biology Grade if subjected to the unit definition assay used for Thermolabile Proteinase K? A: Proteinase K, Molecular Biology Grade yields an activity concentration of 800 U/mL using the traditional denatured hemoglobin activity assay. The same sample yields an activity concentration of ~4,000 U/mL using the more modern nitroanilide substrate visible absorbance activity assay that is used for Thermolabile Proteinase K.