Objectives
Conduct Bradford analysis on protease (trypsin) degraded samples using florescence.
Procedure
- 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.
- We weighed out 7 eppendorf tubes of dried gold fibers.
- We added 978.4 µL 50mM Tris buffer 10 mM CaCl2 at pH 8 to each of the 7 dried gold fiber tubes.
- 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.
- We added 21.57 µL of Trypsin solution to each gold fiber and buffer solutions.
- We placed the 7 tubes of gold trypsin and buffer into a 37C water bath.
- We started the timer.
- While the samples were heated we prepared Bradford assay in plastic cuvettes. We did a 1 to 4 dilution of Bradford to Tris buffer.
- Then we added the following to each cuvette.
- 600 uL of diluted Bradford
- 1650 uL of buffer
- 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.
- Then we ran the sample on the UV/Vis spectrophotometer, recorded and saved the data.
- We did steps 10-11 for all 7 samples.
Data
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.
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