Technical Tip: Performing Assays with Reduced Sample Volumes and Improved Sensitivity in
Tilted-Bottom 384-Well Microplates

Krista Witte, Ph. D., Sr. Director of Chemistry and Consumables R&D

The 384TW microplate is a new 384-well, tilted-bottom, black, polypropylene microplate whose sloped well bottom has been specifically designed to provide benefits not provided by standard 384-well microplates on the Octet RED384 and Octet QK384 systems:

• 3- to 5-fold reduction in required sample volume - only 40 µL for both quantitation and kinetic applications
• Reduction in the interference from light reflecting off the bottom of the plate reduces background noise for high-sensitivity applications such as small molecule kinetics or fragment screening
• The 384TW microplate conforms to SBS standard dimensions and is compatible with standard laboratory robotics systems. It can be filled using standard multichannel pipettes and automated liquid handlers.

Instructions for Use

The 384TW is compatible with the Octet RED384 and Octet QK384 systems. Assays are set up and run as one would in flat-bottom 384 well microplates with the following exceptions:

• Minimum sample volume is 40 µL. Maximum sample volume is 100 µL.
• Care should be taken when pipetting solution into the wells to prevent air bubbles from being trapped in the wells - bubbles can result in spikes in the assay data.
• Set up the assay with the sensor offset at 3 mm. For more information on Sensor offsets and how to set them, please refer to the Octet 384 System Data Acquisition User Guide.
• Set the “Delay experiment start” option in the Octet Data Acquisition software to a minimum of 10 minutes (600 seconds) to allow the plate to equilibrate inside the instrument prior to the first data point.
• Always check the “Shake sample plate while waiting” option in the Data Acquisition software. Shaking the plate during equilibration helps eliminate bubbles from the wells.

Performing Assays Using Limited Sample Volume

When assaying precious samples, the ability to limit the volume of sample per experiment is crucial. The 384TW enables sample volumes as low as 40 µL per well. This is a 3-fold decrease from the volume required in a standard 384-well microplate and a 5-fold decrease from the sample volume required in a 96-well microplate. The assay performance (dynamic range, precision, accuracy) of both kinetic and quantitation assays in tilted-bottom plates are equivalent to those in standard flat-bottom plates, as shown in the figures below.

Improved Sensitivity in Small Molecule and Peptide Analysis

In high-sensitivity screening and kinetics applications, the limit of detection (smallest significant signal) is often set at 3X the standard deviation of the baseline signal. The 384TW decreases Octet RED384/Octet QK384 system artifacts, decreasing the variation (and thus the standard deviation) of the baseline signal, enabling smaller signals to be resolved from the baseline noise.

Table 2 contains data from Octet RED384 using Super Streptavidin (SSA) biosensors. Data shown includes the average signal and standard deviation of the baseline of SSA biosensors in PBS. Averages and standard deviations were calculated from 1920 replicates each. The lower signal limit of detection shown is defined as 3 times the standard deviation over the average baseline. For many screening campaigns this would define the smallest signal that could be considered a positive binding sample. In this experiment the 384TW allows for the detection of approximately 2-fold lower signal than would be possible in the standard 384-well flat-bottom plate.

For more information on the 384TW microplate, please visit www.fortebio.com.

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