Services

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

  1. Buffer composition
  2. pH
  3. Temperature
  4. Ionic strength
  5. Osmolarity
  6. Monovalent ions (Na +, K +, C1 -)
  7. Divalent cations(Mn 2+, Mg 2+, Ca 2+, Zn 2+, Cu2+, Co 2+)
  8. Reological modulators (glycerol, polyethyleneimine glycol [PEG])
  9. Polycations (heparin, dextran)
  10. Carrier-proteins (bovine serum albumin [BSA], casein)
  11. Chelating agents (ethylene diamine tetraacetic acid [EDTA}, ethylene glyrol tetraacetic acid [EGTA])
  12. Blocking agents (polyethyleneimine [PEI], milk powder)
  13. Reducinig agents (dithiothreitol [DTT], ß-mercaptoethanol)
  14. Protease inhibitors (phenylmethylsulphonyl fluoride [PMSF], leupeptin)
  15. Detergents (Triton, Tween, CHAPS)

top

Facility | Services | Resources | FAQ | Contact Us | My ChemCORE | News

Copyright © 2005 Johns Hopkins University. All Rights Reserved. Site designed by Academic Web Pages