User:Roberta Diaz Jimenez/Notebook/CHEM 471 Experimental Lab/2015/09/29

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Objective

Perform a Bradford Analysis to analyze the degradation of protease trypsin.

Procedure

In order to do the Bradford Assay of Protease Degradation, we followed Dr. Hartings' protocol, and did the following specific steps:

  • Protease Sample Prep.
  1. Used eppendorf tube no. 1 that weighed 1.0269g, and contained 0.00108g.
  2. Added 1mL of phosphate/CaCl2 buffer.
  3. Final concentration: 46.35µM
  • Sample Prep.
  1. Used 7 eppendorf tubes, each containing gold fibers.
  2. To each tube add:
    1. 0.9784mL of buffer
    2. 0.02157mL of trypsin (add this at the time of putting the tubes in the hot water bath).
  • Blank Prep.
  1. In on eppendorf tube add:
    1. 0.9784mL of buffer
    2. 0.02157mL of trypsin
  • Bradford Dilution
  1. Dilute 1mL of Bradford in 3mL of buffer such that the resulting solution is 1:4.

Calculations:

   V1 = [(1µM)(1mL)]/46.35µM = 0.02157mL, amount of trypsin solution needed
   Volume of buffer: 1mL - 0.02157mL = 0.9784mL

Data

Graph 1. Image:BradfordGraph1Sept29.png Graph 2. Image:BradfordGraph2Sept29.png


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