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Tutorial
Popular Assay Technologies
Assay
Technology |
Limitations* |
Fluorescence |
Inner
filter effect at high
concentrations of fluorophore
(usually 1µM) |
Fluorescence
polarization |
Usually
30% substrate depletion
required |
Capture
techniques
(ELISA, SPA,
FlashPlate, BET,
others |
Concentrations
of the reactant captured
must be in alignment
with the upper limit
binding capacity. |
Capture
techniques and anyone
monitoring binding |
Nonspecific
binding(NSB) of the product
or aof any reactant to
the capture element (bead,
plate, membrane, antibody,
etc.) may result in misleading
activity determinations. |
|
Sensitivity
limits impose lower limit
to the amount of product
detected |
*These limitations also apply to ligand in binding
assays or other components is assay’s monitoring
any kind of binding event
Assay Formats for Various Target Types
Assay
formats |
Target
Type |
Biomedical |
Cell-based |
GPCRs |
|
FLIPR,
reporter gene melanophores |
Ion
channels |
|
FLIPR,
VIPR |
Nuclear
hormone receptor |
FP,
TRET, SPA |
Reporter
gene |
Kinases |
FP,
TRET, SPA |
|
Protease |
FLINT,
FRET, FP, SPA |
Reporter
gene |
Other
enzymes |
FLINT,
FRET, FP, SPA,TRET,colorimetry |
|
Protein-protein |
TRET,
BET, SPA |
Reporter
gene |
Factors to consider for optimizing an In vitro assay
- Buffer composition
- pH
- Temperature
- Ionic strength
- Osmolarity
- Monovalent ions (Na +,
K +, C1 -)
- Divalent cations(Mn 2+,
Mg 2+, Ca 2+, Zn 2+, Cu2+,
Co 2+)
- Reological modulators (glycerol,
polyethyleneimine glycol
[PEG])
- Polycations (heparin, dextran)
- Carrier-proteins (bovine
serum albumin [BSA], casein)
- Chelating agents (ethylene
diamine tetraacetic acid
[EDTA}, ethylene glyrol tetraacetic
acid [EGTA])
- Blocking agents (polyethyleneimine
[PEI], milk powder)
- Reducinig agents (dithiothreitol
[DTT], ß-mercaptoethanol)
- Protease inhibitors (phenylmethylsulphonyl
fluoride [PMSF],
leupeptin)
- Detergents (Triton, Tween, CHAPS)
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