Objective
- Conduct Bradford Analysis between 10 nM Proteinase K and AuNP fibers
Description
The detailed protocol can be found in this notebook entry by Dr. Hartings.
- AuNP Fibers Preparation
- 7 eppendorf tubes with AuNP were centrifuged at 1500RPM for 1 min.
- Their supernatant was aspirated
- Protinase K 1 μM Preparation
- Calculations
- (0.00121g)(1mol/28900g)(1/0.001L)=41868 nM Proteinase K
- M1V1 = M2V2 => (41868 nM)*(V1) = (10nM)*(1mL) => V1 = 0.238843 uL
- Dilution to 10nm
- M1V1 = M2V2 => (41868 nM)*(0.1mL) = (x)*(10mL) => M2 = 418.685 nm
- M1V1 = M2V2 => (418.68 nM)*(V1) = (10nM)*(1mL) => V1 = 23.9 uL (976.1 uL of buffer needed to make 1mL)
- Incubation
- 14 samples (7 with fibers, 7 blanks without fibers) were incubated in a 37°C shaking water bath for 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hrs, 1.5 hrs, and 2 hrs each.
- Bradford Analysis
- At each time point, the samples were taken out of the water bath and centrifuged at 13500 RPM for 1 minute.
- 750 μL of each sample and blank was placed in a plastic cuvette with 600 μL of pre-mixed Bradford dilution and 1650 μL of buffer.
- Samples were run from 400nm to 800nm on UV-Vis.
Results
This graph shows the absorbance of the 10nm Proteinase K samples from 400nm to 800nm. These are corrected with their blanks.
This is the peak absorption vs time curve of 10nm Proteinase K digestion.
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