To analyze the digestion of Proteinase K at 1 μM using fluorescence assay
- Prepping Fiber Samples
- 7 fiber samples were obtained and spun at 300 RPM for 10 min
- The supernatant was removed
- Proteinase K tube 1B was mixed with 1 mL of 50 mM Tris/10 mM CaCl2 pH 8 buffer
- Proteinase K concentration: (0.00166g)*(1mol/28,900g)*(1/0.001L)= 0.0000574 M Proteinase K
- Amount of Proteinase K solution needed for 1mL with 1μM concentration: M1*V1 = M2*V2 => (57.4 μM)*(V1) = (100 nM)*(1 mL) => V1 = 0.017 mL
- Amount of Buffer solution need to get to 1mL: (1mL total)-(0.017 mL Proteinase K solution) = 0.983 mL buffer
- Incubating Samples
- 17 μL Proteinase K and 983 μL buffer were mixed in the 7 fiber tubes as well as 7 blank eppendorf tubes
- Eppendorf tubes were incubated on a shaker in a 37°C water bath for 10 min, 15 min, 30 min, 45 min, 1 hrs, 1.5 hrs, and 2 hrs for one of the fiber tubes and one of the blanks.
- Running Samples
- Each sample was centrifuged at 12000 rpm for 1 min to collect the remaining fibers
- For each sample/blank, 20uL was taken and placed in a 600uL eppendorf tube along with 140uL of Assay Buffer and 40uL of Assay Reagent.
- Taking Measurement
- Excitation at 390 nm
- Emission from 400 to 650 nm
This graph shows the fluorescence curves for the samples and their respective blanks.
This graph shows the concentration of 1uM proteinase K n mg/ml over time using the calibration curve for the proteinase K with lysozyme.