Ensuring Assay Success: The Right Plate Makes All The Difference

Transcreener® HTS Assays rely on direct immunodetection of nucleotides with a far-red readout in a simple mix-and-read format.  The assays are available in three different detection modes, fluorescence polarization (FP), fluorescence intensity (FI), and time-resolved Forster resonance energy transfer (TR-FRET).  A good microplate reader and the right choice of plates to differentiate specific signal versus background signal or “noise” is crucial to the success of these assays.  These simple guidelines will help you select the right type of assay plate and ensure that you succeed with Transcreener.

Key Factors:

1. Black vs. White

The primary difference between white and black assay plates is their reflective properties. White plates reflect light and will maximize light output signal; black plates absorb light and reduce background and well-to-well crosstalk.   

When Running FI & FP Assays, Use Black Plates

FI and FP assays utilize either red or far-red fluorophores: Alexa 594 or Alexa 633. These fluorophores have relatively short half-lives. We recommend running assays utilizing short half-life fluorophores in black plates to reduce background autofluorescence.  Far-red tracers using excitation wavelengths around 630 nm usually cause less autofluorescence than wavelengths in the UV/Vis range.   Autofluorescence is fluorescence resulting from substances other than the fluorophore-of-interest, and can negatively affect an assay by increasing background signal. Many components of assays, including buffers and biological samples, can autofluorescence.  Autofluorescence is triggered by the same excitation light used to excite the fluorophore in the fluorescence assay.

When Running TR-FRET Assays, Use White Plates

Time-resolved fluorescence assays involve fluorophores that have longer half-lives. Transcreener TR-FRET assays use Terbium chelates. Because of the longer half-life of the fluorescent signal, you can set up your instrument to incorporate a “lag time” or “delay time” between the time the fluorophore is excited, and the time you begin reading the emission signal (time-resolved mode). This allows background autofluorescence to fade before you begin collecting emission signal from assays involving a long half-life fluorophore. Because of this, time-resolved fluorescence assays give a better signal when run in white plates. A white assay plate will result in higher raw signals, because the light is reflected maximally by the white color of the plate. The use of black plates will result in lower raw signals, because the black color of the plate can quench the light.

2. Type of plastic and surface coating

The type of plastic used is critical. Some plasticizers that may leach into the solution can auto-fluoresce. Other factors to keep in mind are the stability of the plates at -20˚C or -80˚C when storing dispensed compounds. For these reasons, we strongly recommend using polystyrene plates. It is also important to use “non-binding” plates that have been treated with a hydrophilic polymer to prevent ionic and hydrophobic interactions to reduce or prevent non-specific adsorptions of the fluorescent labeled substrates or proteins to the microplates.  Care should be taken that the proteins are not binding to the plates. Some types of plates require pre-wetting before performing the assay.

3. Well shape and volume

For 384 and 1536 well plates, respectively, we recommend a 20 μl and 8 μl final volumes, after addition of detection reagents, with round bottoms for efficient mixing.  For 384, a low volume assay plate is necessary to allow sufficient liquid height for instrument sensors with the 20 μl volume.  For researchers using 96-well plates, we recommend final volumes of 50 μl in Half-Area microplates, which are designed to facilitate pipetting of low assay volumes in 96 well format.

Visit Our Assay Plates Page to Learn More

The table below shows the recommended plate types for the three different applications: