Assay Design Flashcards
Why would a direct measurement be used instead of quenching methods?
Non-invasive
Fewer steps, so less errors and better signal to noise
Why would quenching methods be used over direct methods?
The reaction is arrested at different timepoints. An instrument can be used to quantify the chemicals
What factors should be considered when designing an assay?
Range of applicability
Time scale
Sensitivity
Availability
Accuracy/reliability
Expense
Describe spectrophotometry
A light beam passes through a prism and a monochromator selects wavelengths of interest
A photomultiplier detects wavelengths of interest, converting photons to electrical signals
Describe fluorescence
Property of compounds that absorb light at one wavelength and emit at another.
When detected for direct measurement, the setup is like spectrophotometry, but the detector is 90° from the cuvette
When is a pH meter used for observing reactions directly?
When working in the absence of a buffer.
Finding appearence/disappearence if a compound
Describe surface plasmon resonance (SPR)
Label-free instrument that determines Kd for a ligand
Although the values for Kon and Koff are innacurate, the ratios are fine
Binding is measured as a change in refractive index of the surface ligands are attached to.
Describe continuous flow methods
For measuring reactions with a dead time >10^-6 seconds
A pneumatically driven ram pushed two liquids into a capillary tube. A platinum ball acts as a mixer, making it fast
Light is shone on the observation cell
Describe the stopped flow method
Dead time is 10^-3 seconds
Wavelengths are selected by monochromators when detecting the reaction. There is a stopping syringe to halt the reaction.
If left for too long enzymes move back out of the reaction tube
Describe equilibrium perturbation
Uses infrared lasers to cause rapid changes in temperature up an order of 5-10 times
Dead time of 10^-8 - 10^-9 seconds
Describe flash photolysis
Uses lasers to break chemical bonds and trigger a reaction (10^-3)
This can cause dissocation of CO from haemoglobin/myoglobin
Can break bonds in caged ATP to prevent it reacting
Describe quenched flow
Dead time 10^-3
2 solutions are pushed into a delay loop and then the quencher is added.
This is collected in a tube for analysis with radioactivity
How is radiation detected in assays?
Overall radiation does not change
A scintillation counter uses a chemical to transfer radiation to light quanta
High performance liquid chromatography
A column matrix aid separation with pressure. The compound binds to the matrix.
Detected with absorbance and fluorescence
Useful when optical signals cannot be differentiated
How can chemicals without optical signals be measured in HPLC?
Refractive index detectors find elution time from and amount of each sample
