Automated electrophoresis systems integrate the process of separation, detection, and data analysis of proteins or nucleic acids in a single platform and remove possible manual errors during the multifarious steps of the electrophoresis process. Using much smaller sample and reagent quantities and less hands-on time than standard analysis methods, automated electrophoresis systems like Bio-Rad's Experion™ automated electrophoresis system can be used in applications such as purity and yield assessment in protein purification and protocol optimization workflows, RNA integrity assessments before RT-qPCR or next generation sequencing, or separation and detection of DNA fragments from PCR reactions or RFLP analysis
This section describes the Experion automated electrophoresis system from Bio-Rad and how it can be used in the automated electrophoresis workflow. To learn more about the technology behind the Experion system, visit our Experion automated electrophoresis system section.
The Experion automated electrophoresis system employs LabChip microfluidic technology to automate protein and nucleic acid electrophoresis. Using much smaller sample and reagent quantities than standard analysis methods, the Experion system accomplishes analysis in a single 30–40 minute, automated step.
The Experion system includes: 1) automated electrophoresis station, 2) priming station, 3) vortex station used for nucleic acid analysis only, 4) system operation and data analysis tools (software), and 5) analysis kits, which include the (a) chips and (b) reagents for protein (Pro260 kit), standard-sensitivity RNA (StdSens kit), high-sensitivity RNA (HighSens kit), and DNA (DNA 1K and 12K kits) analyses.
- Protein analysis — quantitation and size determination (sizing) of protein samples, useful for quality control, protein purity and stability analysis, protocol optimization, and evaluation of recombinant protein expression
- RNA analysis — evaluation of RNA integrity and concentration. The system automatically generates the RNA quality indicator (RQI), a quantitative integrity assessment, in addition to electropherogram (fluorescence vs. time graph) data, ribosomal RNA ratios, and concentration data. The resulting data makes Experion RNA assays useful upstream of applications such as quantitative real-time PCR (qPCR), microarray analysis, sequencing, and reverse-transcription qPCR (RT-qPCR)
- DNA analysis — quantitation and sizing of restriction digests, amplified DNA, microsatellites, and amplified fragment length polymorphisms (AFLPs)
Sample analysis involves the following steps:
- Preparing the chip (priming, loading, and vortexing) — priming fills the microchannels of the microfluidic chip with the gel-stain solution, which contains both the sieving matrix and fluorescent dye. Samples prepared in Experion sample buffer are then loaded in the sample wells. The chip is vortexed for RNA and DNA analyses
- Running the chip — the chip is inserted into the electrophoresis station, and as the electrophoresis station lid is closed, electrodes come in contact with the solution in the wells. The voltage applied to the sample wells causes the charged molecules to migrate into the separation channel. In the separation channel, the gel-stain solution causes the molecules to move at different rates, depending on their size. During separation, the fluorescent dye, which is specific to the analysis being performed, associates with the lithium dodecyl sulfate (LDS) coating the proteins (protein analyses) or intercalates between the bases of RNA (RNA analyses) or DNA (DNA analyses)
- Detecting the molecules — as the molecules migrate toward the end of the separation channel, a laser excites the dye, causing it to fluoresce if it is bound to the protein-LDS micelle complexes or RNA/DNA fragments. A photodiode detects the fluorescence, and Experion software plots the fluorescence intensity as a function of time, producing an electropherogram. A virtual gel image is generated from the electropherogram data
- Data analysis — once separation is complete, the software subtracts background noise, identifies and integrates peaks, and assigns their sizes and concentrations; the results of data analysis are automatically tabulated and presented in a table. Experion software displays all three forms of data simultaneously (see figure below)
Protein, RNA, or DNA analysis with the Experion system is achieved with various analysis kits, which supply the microfluidic chips, reagents, and instructions required to separate and analyze 10–260 kD proteins (under denaturing conditions), total or mRNA, and double-stranded DNA.
The Experion system can be used for protein applications such as:
- Protein quality control (i.e. monitoring degradation or stability of proteins)
- General protein or antibody purity analysis
- Evaluation of protein expression experiments
- Sample prep protocol optimization
- Analysis of chromatography fractions
See Protein Analysis Using the Experion System for more information.
The Experion system can be used for nucleic applications such as:
- RNA integrity assessment and quality control for qPCR/RT-qPCR, microarray, sequencing
- PCR amplicon confirmation and analysis
- Restriction digest analysis (RFLP)
- mRNA integrity and size distribution assessment
See Nucleic Acid Analysis Using the Experion System for more information.
The four part training videos presented here provides information on the Experion automated electrophoresis system, helps you refine your chip loading techniques, understand software features and shows an example of a succesful RNA run.
This webinar by Field Application Specialist Sean Taylor presents the advanced features of the Experion software for accurately calculating protein concentrations by using customizable internal standard and standard curve calibration samples.