User:Matthew R Skorski/Notebook/471 - Exp BioChem/2015/10/13

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Objective The objective of today was to run the Bradford Analysis of Protease Degradations for 100 nM Protinase K. Description The protocol is identical to the one used on 9/29/15 except the fibers were spun down at 300 rpm for 10 minutes, not 1500 rpm for 1 minute. Prepping AuNP Fiber Samples 7 AuNP fiber samples were spun at 300 RPM for 10 minutes The supernatant was removed but the fibers were retained Protinase K tube 1C was mixed with 1 mL of 50 mM Tris and 10 mM CaCl2 pH 8 buffer Protinase K concentration: (0.00127g)*(1mol/28,900g)*(1/0.001L)= 0.0000440 M Protinase K Amount of Proteinase K solution needed for 1mL with 100 nM concentration: M1*V1 = M2*V2 => (440000 nM)*(V1) = (100 nM)*(1 mL) => V1 = 0.002 mL Amount of Buffer solution need to get to 1mL: (1mL total)-(0.002 mL Protinase K solution) = 0.998 mL buffer Incubating Samples 2 μL Protinase K and 998 μL buffer were mixed in the fiber tubes as well as blank eppendorf tubes Eppendorf tubes were incubated on a shaker in a 37°C water bath for 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hrs, 1.5 hrs, and 2 hrs for one of the fiber tubes and one of the blanks. Running Samples Once the time for a sample was up the tube was centrifuged at 300 rpm to collect the remaining fibers To a plastic cuvette was mixed in 600 uL of pre-mixed Bradford dilution, 750 uL of sample, and 1650 uL of buffer Samples were run from 400-800 nm on the UV-VIS Results The graph below shows the absorbance of the samples as function of the wavelength of incident light when corrected. The six samples were first corrected for by subtracting the absorbance of the superblank of buffer and Bradford reagents. Then the samples were corrected for again by subtracting the results of the first correction by the blanks for their corresponding time periods. Then, the isosbestic point at 535 nm from each samples was subtracted from all the wavelengths of that sample. The graph below shows the peak for the absorbance at 600 nm. The graph does not appear to follow any linear trend. It has a max at 60 minutes but dips at 30 and 90 minutes. The trial should be re-run to ensure that the trend is accurate and not due to human error.