L11 An introduction to assays

What is an assay

IC₅₀, EC₅₀, K_i, K_d

Ideally you want these numbers to be small as possible

What are PAINS

give a couple common examples

Molecules often detected in assays that are active owing to artefacts or lack of specificity. (Reaction with amino acids that are non-specific or interference with the assay mechanism)

Enzyme kinetics - K_m and k_cat use in tandem with assays

What equation relates K_i to IC₅₀ and why is this equation useful

Using these studies and an assay you can find the paramaters necessary to calculate K_i

Converting between K_i and IC₅₀ may be necessary depending on the standard used by the lab/paper.

Competitive inhibition - How does K_m and V_max change with increasing inhibitor

K_m increases with inhibition as binding affinity has effectinvely dropped. V_max is unaffected as a large substrate concentration is gonna outcompete the inhibitor

Non-competitive inhibition - How does K_m and V_max change with increasing inhibitor

K_m is unaffected by inhibition as the binding affinity to intact enzymes remains constant but V_max decreases with inhibition

What is uncompetitive inhibition and how is K_m and V_max affected

Both K_m and V_max decrease with inhibition. K_m decreases as [ES] is decreased as [ESI] is formed

How can we assess the quality of an assay mathematically

The Z’ factor - measures signal to noise and signal to background.

Microplate assay features - what does the detergent do

Dtergent ensures the compoents are not bound to the sides of the plate

Advantages and disadvantages of microplate assasy

Well-established, familiar process with instruments that are readily available at most labs and standardised.

The process can also be automated by robots