Assay Design

Question 1

What are the 2 types of assay design methods?

Answer 1

Direct
Quenched (Indirect)

Question 2

Characteristics of Direct methods? (5 characteristics)
What is rare and limited in direct methods? (2 things)

Answer 2

Reaction observed as it happens
Non-invasive (nothing added to arrest reaction)
Reaction is probed using techniques that are specific to chemical of interest
Simpler and quicker than quenching
Fewer steps means less chance of error

Rare to be able to monitor all reaction components simultaneously
Types of reactions that can be measured directly is limited

Question 3

Characteristics of Quenching methods? (3 characteristics)
Drawbacks? (1 drawback)

Answer 3

Reaction is arrested and the extent of the reaction is assessed at each timepoint
Reaction arrested by quenching agent
Allows for measurement of multiple components; Less limited than direct

More error prone to due to more steps

Question 4

3 steps of Quenching?
- How many sets of instruments per step?

Answer 4

1. Starting (triggering) and stopping (quenching) the reaction
2. Separation of substrates and products
3. Quantification and Identification

• One set of instruments per step

Question 5

What is essential for reactions which occur very fast?
- What is used for this?

Answer 5

Must be mixed very fast
- Cannot be done manually so special instruments are used for fast mixing

Question 6

If a reaction cannot be mixed fast enough what methods are used?
- Describe this
What is an example of these methods? (hint - heat/pressure)

Answer 6

Equilibrium perturbation methods
- This doesn’t mix 2 solutions but instead relies on other factors to trigger reaction

Components mixed at low temperature which causes equilibrium position to not favour reaction
Upon heating, equilibrium shifts to new position and reaction is triggered
- Pressure change is another example

Question 7

What is HPLC?
What is it for?
How is it so accurate?

Answer 7

High Performance Liquid Chromatography

Separates small molecules (e.g. protein)

State-of-the-art separation technology directly upstream of a wide variety of useful detectors - Increased accuracy

Question 8

Steps of Quenching Assay in more detail:
Step 1 - Stopping (Quenching) reaction (1 detail)

Answer 8

Done by adding a quenching agent (see Table 3)

Question 9

Steps of Quenching Assay in more detail:
Step 2 - Separating Reaction Components (2 details)

Answer 9

Some old techniques are preferred
However HPLC is a modern favourite

Question 10

Steps of Quenching Assay in more detail:
Step 3 - Quantifying and Identifying components (1 detail)
- What is the signal of choice for sensitivity?

Answer 10

Once separated, different components can be quantified whether they have specific signals or not
- Radioactivity is the signal of choice where sensitivity is required
- Look at Table 1 for methods of identification

Question 11

What molecules is NMR very sensitive too?

Answer 11

Hydrogen
Nitrogen
Carbon

Used for simple quantitative measurements of small organic compounds as well as highly complex determination of protein structures

Question 12

What are X-Rays used for?

Answer 12

Studying structure of macromolecules in crystals

Question 13

How is absorbance measured with a direct method?
Beers law equation?
What can the observed absorbance equation be used for?

Answer 13

Wavelength of light selected using a “monochromator”
This incident light (Io) is shined onto sample and the transmitted light (I) is detected using a “photomultiplier”

Beers Law - log(Io/I)

Observed absorbance equation tells us that the absorbance is directly proportional to the concentration of absorbing compound

Question 14

How does a Fluorometer measure fluorescence?
Set-up?
What is fluorescence proportional to?

Answer 14

Similar set up to absorbance spectrophotometer
Different as:
- Light is transmitted at 90° instead of 180°
- Light goes through 2nd “monochromator”

Fluorescence is proportional to concentration

Question 15

What is meant by SPR (Surface Plasmon Resonance) being “label-free”?
What does it measure?
How does it work?
- Refractive index?
How is it good in pharmocology?

Answer 15

Doesn’t require reaction to have an optical signal

Measures Kd for ligand binding to a protein

Solution of protein flows over solid surface with ligand attached
Binding is measured as change in refractive index; Property of compound to reflect off light

Good at suggesting which drugs bind tightest

Question 16

What is the dead-time of an apparatus?
- What does dead-time of 10us mean?

Answer 16

The timescale of reaction an apparatus could measure
- Dead-time of 10us means it can measure reactions with a half-time of 10us or more

Question 17

Principle of continuous flow? (3 points)

Answer 17

Push 2 liquids into a small capillary using a pressure driven ram at a constant rate
Pushed into observation cell where the liquids are mixed to trigger reaction
Light is shone on the observation cell and data is collected

Question 18

How is stopped-flow different to continuous flow?

Answer 18

Has a stopping syringe which regulates amount of enzyme and substrate in observation cell; Can stop reaction at a certain point

Question 19

In flash proteolysis, what can infra red lasers be used to do?

Answer 19

Break specific chemical bonds in compounds such as “caged-ATP”
Releases ATP into solution and a reaction is triggered

Question 20

Quenched-flow is similar to stopped-flow. How are they different?

Answer 20

2 solutions pushed into delay-loop, and then after a specified time, a 3rd solution (quencher) is mixed in, stopping reaction

Question 21

Why is a scintillant used when measuring radioactivity?

Answer 21

Converts radiation energy into light energy which can be quantified

Question 22

When is HPLC useful?

Answer 22

When reactants and products have optical signal and can only be differentiated once separated

Question 23

What does each peak in a HPLC chromatogram mean?

Answer 23

Area of each peak gives measure of amount of each constituent in sample