Bio-Rad Experion Protein Analysis Kits - Manual (Page 52)

Appendix A: How the Experion ™ System Works
The Experion automated electrophoresis system performs electrophoresis of samples within a
microfluidic chip. Within each chip, a series of microchannels connects the sample wells to a separation
channel and buffer wells. A set of electrodes in the electrophoresis station applies a voltage across
the microchannels, causing charged molecules in the samples to migrate into and through the
separation channel. Samples are run sequentially, with a sufficient lag between them to prevent cross-
contamination. For separation, the microchannels are filled with a proprietary gel-stain solution (GS)
that acts as a sieving matrix, and under denaturing conditions in the presence of lithium dodecyl sulfate
(LDS), the sample proteins migrate through the separation channel at a rate based on their size. Finally,
proteins interact with a fluorescent dye during separation and are detected as they pass a laser and
photodiode detector (laser-induced fluorescence).
Protein analysis is accomplished with the Experion Pro260 analysis kit and involves the following steps:
n
Preparing the chip (priming and loading) — Priming fills the microchannels of the
microfluidic chip with GS, which contains both the sieving matrix and fluorescent dye.
Protein samples are then prepared in Pro260 sample buffer, which contains LDS, heat-
denatured, and added to the sample wells
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Running the chip — The chip is inserted into the electrophoresis station, and as the
instrument lid is closed, electrodes come into contact with the solution in the wells. Voltage
is applied to the sample wells of the chip, causing the charged, LDS-coated protein ions
to migrate into the separation channel. In the separation channel, the different proteins
separate as they move at different rates through the gel matrix, depending on their size.
During separation, the fluorescent dye associates with the LDS micelles coating the
proteins and with free micelles
1
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Detecting the fragments — As the molecules migrate toward the end of the separation
channel, destaining occurs. Pro260 gel (G), which does not contain dye or LDS, flows
alongside the separation channel. Diffusion of free LDS into this detergent-free zone
reduces the concentration of LDS below its critical micellar concentration, releasing dye
molecules from unbound micelles. When it is free of the hydrophobic interior of the LDS
micelle, the dye fluoresces weakly; the background signal is thus reduced. Dye-micelle
complexes are more stable to the destaining process when they are bound to proteins.
Downstream a laser excites the dye, causing it to fluoresce if it is bound to the LDS micelle-
protein complexes. A photodiode detects the fluorescence, and Experion software plots the
fluorescence intensity vs. time to produce an electropherogram and a virtual gel image
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Analyzing the data — Following separation, Experion software subtracts background noise,
removes spikes, identifies and integrates peaks, and assigns their sizes and concentrations.
Following analysis, parameters may be changed and the data reanalyzed
1 An important difference between the Experion system and traditional sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) lies in how sample fragments are detected: in SDS-PAGE, samples are generally stained in the gel
once separation is completed, while in the Experion system proteins are stained with a fluorescent dye during separation.
48 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Experion Automated Electrophoresis System