User:M Jaffe/Notebook/Maxis Protease Lab 471/2015/10/07: Difference between revisions
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== | |||
==Procedure== | |||
Bradford Analysis of Protease Degradation | |||
You should make samples for measurements at 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hr, 1.5 hr, 2 hr. (Note, this is for the first time you do this. As you get better, I will expect you to get more measurements done). | |||
# Protease Prep | |||
## Place the 1mL of Tris/CaCl2 buffer in the pre-measured protease sample and record the concentration | |||
# Sample Prep | |||
## To a fiber sample tube (that has been centrifuge and had the supernatant removed) add the appropriate amounts of buffer and protease | |||
### The total volume should be 1mL | |||
### The final protease concentration should be 1uM | |||
### Vortex the sample to disperse the fibers | |||
# Blank Prep (you will have one blank to match each sample) | |||
## To a clean eppendorf tube add the appropriate amounts of buffer and protease | |||
### The total volume should be 1mL | |||
### The final protease concentration should be 1uM | |||
# Incubate Sample and Blanks | |||
## Place the tubes (for both the Blanks and Samples) in the shaker with the 37C water bath | |||
# Measurement | |||
## Remove the tubes (the sample and a corresponding blank) from the water bath at the appropriate time | |||
## For the case of a sample with fibers, centrifuge the sample for 1 min to pull any fibers to the bottom of the tube | |||
## To a plastic cuvette add | |||
### 600 uL of pre-mixed Bradford dilution (that I give you) | |||
### 750 uL of sample | |||
### 1650 uL of buffer | |||
## Record the UV-Vis spectrum from 400-800 nm | |||
# Using the Bradford standardization curve and your data from the 0 fiber sample, determine the concentration of released protein in your solution. | |||
Latest revision as of 01:14, 27 September 2017
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ProcedureBradford Analysis of Protease Degradation You should make samples for measurements at 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hr, 1.5 hr, 2 hr. (Note, this is for the first time you do this. As you get better, I will expect you to get more measurements done).
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