Western Blot Trf Read Mode - Molecular Devices SpectraMax Paradigm User Manual

Western Blot TRF Read Mode

Protein detection is an important task for pharmaceutical and clinical research, and Western
Blots (WB), or protein immunoblots, are one of the most common methods employed for
this purpose. Various techniques are used to detect proteins on Western Blot membranes
including fluorescence, silver staining, and chemiluminescence. However, each technique has
its limitations, and there is a continuous need to improve quantitation, accuracy, and
dynamic range of Western Blots.
The Molecular Devices ScanLater™ Western Blot Assay Kit is a novel system for protein
analysis that is incorporated into a SpectraMax Paradigm Multi-Mode Detection Platform.
Membranes are incubated with Eu-chelate labeled secondary antibodies or streptavidin that
bind specifically to the target protein-specific primary antibody. Europium has a long
fluorescence lifetime, on the order of 1 msec, and detection is done in Time Resolved
Fluorescence (TRF) mode which significantly reduces background from auto-fluorescence or
other sources of short lifetime emissions. The membranes are placed into a microplate
reader where they are scanned with the ScanLater Western Blot (WB) Detection Cartridge.
The method does not involve enzyme detection, and the Eu-chelates are resistant to photo-
bleaching, so the signal remains stable for long periods of time (weeks to months). This
permits repeat reading of membranes and the potential for comparison of band intensities
to known standards for more accurate quantitation.
The TRF detection employs photon counting, so the theoretical dynamic range is >10
practice, however, dynamic range is limited by saturation of binding sites on high-abundant
bands and non-specific binding to background membrane. There is also no camera
blooming, as can occur with chemiluminescence or fluorescence detection, so the system
gives sharp bands and excellent image quality.
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Chapter 2: Read Modes and Read Types
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