Bio-Rad Experion Protein Analysis Kits - Manual (Page 31)

Appendix A — How the Experion™ System Works
The Experion 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; therefore,
the sample fragments migrate through the separation channel at a rate based on their size
and charge. Finally, sample fragments interact with a fluorescent dye during separation and
are detected as they pass by a laser and a photodiode detector.
Protein analysis is accomplished with the Experion Pro260 analysis kit and involves the
following steps:
1. Preparing the chip (priming and loading) –– Priming fills the microchannels of the
microfluidic chip with the gel-stain solution, which contains both the sieving matrix and
fluorescent dye. Protein samples are then prepared in Pro260 sample buffer, which
contains lithium dodecyl sulfate (LDS), and are added to the sample wells.
2. Running the chip –– The chip is inserted into the electrophoresis station, and as the
instrument lid is closed, electrodes come in 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
9 .
3. Detecting the fragments –– As the fragments migrate towards the end of the
separation channel, destaining occurs. Pro260 gel (G), which does not contain dye or
LDS, flows alongside the sample. 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 from where destaining occurs, a laser beam 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 versus time to
produce an electropherogram and a virtual gel image.
4. 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.
9 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.
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