Thermal stability biochem lab 2
The goal of this protocol is to determine the temperature that causes the AGPase to denature to the extent that there is loss of enzyme’s activity.
- Make a 1ml dilution of the protein sample that has around 0.8-1 absorbance reading (check your kinetics results from Lab 2 (Week 2) or 3 (Week 3))
- Label 9 microcentrifuge tubes, each with a different temperature: 0 ̊C, 30 ̊C, 40 ̊C, 50 ̊C, 60 ̊C, 65 ̊C, 70 ̊C, 75 ̊C and 80 ̊C
- Add 100 ul of your well-mixed dilution (from step 1) into each of 9 labeled microcentrifuge tubes. Add 10 ul of 10 mg/ml BSA into each tube. Mix well and centrifuge.
- Each tube should be placed at the appropriate temperature for 5 minutes, immediately followed by placing the tube on ice for 5 minutes
- Following heating, all the tubes should be spun in the minispin centrifuge for 10 minutes.
- The supernatants should be carefully removed and placed in a fresh tube.
- Perform an enzyme assay.
- Plot the activity versus the temperature.
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