Jan 21

Question 1

What are the steps in spectroscopic titrations, why is it used?

Answer 1

It measures a change in response based on the current concentration of ligand. As the concentration increases, the change of response will flatten out eventually in a sharp or gradual manner.

1. Put high P conc in sample and blank cuvettes
2. Measure
3. Add samll amount of ligand
4. Measure again
   Repeat 3 and 4

Question 2

What is N that you determine from spectroscopic titrations/

Answer 2

It is the number of ligand binding sites of the macromolecule

Question 3

In spectroscopic titrations, if the binding is weak (no sharp slope to flat transition), how do you still determine N?

Answer 3

You have very concentrated protein to simulate tight binding. Then get the slope to be linear, don’t need saturation, just need the slope

Question 4

How is spectorscopic titrations done with indicators?

Answer 4

Often use Ca2+ as the ligand. We know the Ca2+ and dye kd value, so add dye and protein to solution. Then add Ca2+, can determine how much binds to protein from it.

Question 5

What is total internal reflection fluorescence?

Answer 5

It is when the total internal reflection off the cuvette creates an evanescent wave very close to the glass walls, and it excites the flurophores right by the glass walls. Can obtain high quality images of dyed cells with this method

Question 6

Why use a cuvette with a smaller pathlength?

Answer 6

Limit inner filter effect if turbid sample, if you have a small sample.