User:Asya L. Tucker/Notebook/Asya 571/2015/09/29

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Objectives

Conduct Bradford analysis on protease (trypsin) degraded samples using florescence.

Procedure

  1. We retrieved one of our Trypsin eppendorf tube from our freezer box. We added 1 mL of 50mM Tris buffer 10 mM CaCl2at pH 8 to the tube and recorded the concentration which was 46.35 µM.
  2. We weighed out 7 eppendorf tubes of dried gold fibers.
  3. We added 978.4 µL 50mM Tris buffer 10 mM CaCl2 at pH 8 to each of the 7 dried gold fiber tubes.
  4. We scrapped the gold fibers that were stuck to the sides of the eppendorf tube. We used the vortex on each tube for approximately 1 minute.
  5. We added 21.57 µL of Trypsin solution to each gold fiber and buffer solutions.
  6. We placed the 7 tubes of gold trypsin and buffer into a 37C water bath.
  7. We started the timer.
  8. While the samples were heated we prepared Bradford assay in plastic cuvettes. We did a 1 to 4 dilution of Bradford to Tris buffer.
  9. Then we added the following to each cuvette.
    1. 600 uL of diluted Bradford
    2. 1650 uL of buffer
  10. After each time interval, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hr, 1.5 hr, 2 hr., we removed the Trypsin samples from the water bath, centrifuged the sample for 1 min to pull any fibers to the bottom of the tube and then added 750µL to the already prepared cuvette.
  11. Then we ran the sample on the UV/Vis spectrophotometer, recorded and saved the data.
  12. We did steps 10-11 for all 7 samples.

Data

Graph 1uM Trypsin.Abs vs Wl.png

Graph 1uM Trypsin.Abs vs Time I.png

Graph 1uM Trypsin.Abs vs Time II.png

Results

Using the Bradford standardization curve and your data from the 0 fiber sample, determine the concentration of released protein in your solution.

Notes

We did not take a blank after every measurement so we will run them tomorrow.