Two-Dimensional Gel Electrophoresis
Two-Dimensional Electrophoresis (2DE) is an integral tool in the study of proteomics and is often described as the proteomics "work horse". 2DE enables the separation of complex protein mixtures by combining isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second dimension.
Isoelectric focusing is a sophisticated and reproducible technique where proteins are immobilized at their isoelectric point. Increasingly narrower ranges can be used to focus on particular protein pI ranges, in order to "zoom" into your area of interest.
The 2DE capabilities at APAF include 11cm mini-gel, 17/18cm and 24cm large format gel systems. APAF routinely runs a wide variety of complex samples on these various formats, with the mini-gel systems generally used to obtain an initial overview of a sample’s protein distribution. The large gel systems are then used to analyse and investigate regions of interest with the aim of visualising detailed sections of the proteome. Protein profiles can be visualised using both fluorescent and visible stains.
The EttanTM IPGphor™ 3 (GE Healthcare) isoelectric focussing intruments at APAF and sample-cup loading of IPG strip (Immobilzed pH Gradient)
There are many different stains available for SDS PAGE. A selection of commonly stains used at APAF is listed below. Coomassie is a visible stain where as Sypro Ruby, Deep Purple and CyDyes are fluorescence stains/dyes. Fluorescent stains are recommended for image analysis because of their wide linear dynamic range. The term, "linear dynamic range" refers to the number of orders of magnitude where the detector’s response increases proportionally with protein amount.
Common Stains and Dyes used for 2D SDS PAGE
||Linear dynamic range
|Coomassie Brilliant Blue
|Minimal Cy Dyes
APAF has two Typhoon Trio laser scanners (GE Healthcare) for fluorescence gel image capture.
Summary of the features of the Typhoon Trio:
- 5 orders of detection, i.e., 100 000 shades of grey available
- Red, green and blue excitation lasers
- Powerful excitation source for low abundance spots
- Up to 10µm resolution
APAF also has the Fijifilm LAS-3000 CCD camera for chemiluminescence and gel documentation.
What is 2DE Image Analysis?
Image analysis is extracting tangible data out of the 2DE image with specially designed software.
Depleted human plasma fluorescence gel, IPG: 4-7, second dimension: 8-18%
Typically it involves detecting spots and warping separate images to align like-spots of the same protein. Spot data comes from the level of spot darkness which is proportional to the level of protein staining or dye labeling of particular amino acids.
3D-views of differentially expressing protein spots from Progenesis PG240
Image analysis is a powerful technique which can readily identify differences between gel images. Similarities between gel images can also be elucidated but most queries biologists have concern protein expression differences. Image analysis software can group your samples into separate biological groups such as Control and Diseased and then identify proteins for biomarker candidacy. APAF uses Nonlinear Dynamics’ Progenesis PG240 image analysis software for traditional single stain and DIGE experiments. APAF has recently incorporated Nonlinear Dynamics’ TT900 S2S new powerful image alignment software into their analysis: http://www.nonlinear.com/news/press/2006/1904.asp
Image Analysis of DIGE (Difference Gel Electrophoresis) images
Image analysis of DIGE images is simplified as compared to traditional single stain approaches due to the co-migration of up to 3 samples on one gel. All 3 samples can have the same spot segmentation (spot outline) and will not require warping. Having the same spot segmentation on the 3 channels will also improve spot volume quantitation.
Difference in gel image (DIGE), protein expression differences can be readily identified
If the 3rd sample is replaced with an internal standard (a pool of all your samples) then this can be used to warp images from different physical gels, the ease of warping will be greatly improved as the internal standard image is the same "sample". In this approach, the internal standard is used to pull all the spot data in the experiment together. The internal standard also has a second function which is to increase the reliability of quantitation between gels. Spot volume of your samples will be expressed as a ratio to the internal standard then the ratios can be directly compared to one another.
Statistical Analysis and Experimental Design for Biomarker Discovery
We can never prove anything true, only the opposite to be false
Often researchers are not prepared for the amount of variation in spot data, especially between organisms of the same species and the same biological population. Also, there can often be 5 orders of magnitude in spot volume between your smallest and largest spot, i.e., 1 to 100 000! Depending on the given spot data and the experimental aims, replication should be introduced into your experimental design at the appropriate levels. If there is a high level of seemingly random variation between organisms of the same biological population then the number of samples should be increased. Similarly if there is a high amount of variation between gel replicates, replication should be introduced at the gel level (certain sample types on particular pH ranges are prone to technical variation). Also, equal numbers of samples between your biological groups is desirable. At APAF, technical variation is minimised by our experience in sample preparation, electrophoresis, gel pouring, imaging and image analysis. Customers are welcome to discuss experimental design with us.
Triplicate gels, n=1059 matched spots
The electroblotting methods used by APAF incorporate a semi-dry blotting system that is used to maximise blotting efficiency by incorporating a peltier cooling system that ensures optimal transfer of proteins. This system has been designed in-house to meet the specific needs of large format protein electroblotting systems.
Protein Quantitation Determination
Based on Coommassie™ this assay technique has been trusted for many decades. Compatible with 2D sample buffers and BSA standard produces linear response curve between 50ug – 1000ug.
Fluorescence assay based on FLUOROTECHNICS proprietary fluorophor epicocconone which fluoresces intensely red on binding to proteins. BSA standard produces a linear response curve between 5ug – 500ug. Interfering compounds can be diluted out. For detailed info on FLUOROPROFILE™ visit http://www.fluorotechnics.com