User:Matthew R Skorski/Notebook/471 - Exp BioChem/2015/09/29

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Objective

The objective of today was to run the Bradford Analysis of Protease Degradations for 1 μM Protinase K.

Description

Instructions for the day can be found here.

  1. Prepping AuNP Fiber Samples
    1. 7 AuNP fiber samples were spun at 1500 RPM for 1 minute
    2. The supernatant was removed but the fibers were retained
    3. Protinase K tube __ (think 3) was mixed with 1 mL of 50 mM Tris and 10 mM CaCl2 pH 8 buffer
      1. Protinase K concentration: (0.____g)*(1mol/28,900g)*(1/0.001L)=_____ M Protinase K
      2. Amount of Proteinase K solution needed for 1mL with 1μM concentration: M1*V1 = M2*V2 => (0.0000____)*(V1) = (0.000001)*(0.001) => V1 = 0.0000____L = 0.0____mL
      3. Amount of Buffer solution need to get to 1mL: (1mL total)-(_____mL Protinase K solution) = ______ mL buffer
  2. Incubating Samples
    1. _____Protinase K and _______buffer were mixed in the fibers as well as a blank eppendorf
    2. 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 each.
  3. Running Samples
    1. Once the time for a sample was up the tube was centrifuged at a low speed to collect the remaining fibers
    2. To a plastic cuvette was mixed in 600 uL of pre-mixed Bradford dilution, 750 uL of sample, and 1650 uL of buffer
    3. Samples were run from 400-800 nm on the UV-VIS

Results

As a blank was not run at the same time for each of the timed samples no correction could be made for the data. The blanks were remade on 9/30/2015.



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