Lab 11: PRELAB
PRELAB ASSIGNMENT
- What will you be doing in lab? Why?
- You will need to make a detailed chart of components to add to each well of the 96-well plate. Look back to the chart you made in Lab 9 to remember the important information the chart should contain, and make a blank chart in your notebook that you can use today.
- For a successful DNSA experiment, you want to generate a curve that shows binding of DNSA to HCAII. You want to start at a very low concentration of DNSA, i.e. no binding, and progress to saturation. Sketch what such a curve should look like (label the axes!).
- Similarly, for a successful AZ experiment, you want to generate a curve that shows AZ inhibiting the binding of DNSA to HCAII. Sketch what such a curve should look like (label the axes!).
- You will be asked to choose reasonable highest and lowest concentrations of AZ for your titration. What values will you use?
- Complete planning and calculations for the lab PRIOR to lab!
- You must coordinate with your podmates
- Using the Google Sheets template document as a starting point: Lab 11 Planning Template
- This video from a TA helps explain the planning process:
- Part 1:
- Part 2:
PRELAB QUIZ
Use the BRENDA database again to find published Ki values for acetazolamide inhibition of HCAII. This should give you a general idea for how strongly AZ binds HCAII. Which of the following is the closest approximation?
(Note that in lab 11 you are actually measuring KD, not Ki. However, the Ki should still give you an idea of the strength of binding.)
a) 5 nM
b) 12 nM
c) 12 μM
When running the pilot for part 1 (DNSA), you will test two DNSA concentrations: 20 and 40 μM. What do you expect to see from these test concentrations?
a) They will both give a similar, low fluorescence signal
b) 40 μM will give a much higher fluorescence signal than 20 μM
c) They will both give a similar, high fluorescence signal
Link to the quiz in Canvas: Prelab 11 Quiz