Protein Arrays using Gels
Gel Electrophoresis 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.
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
|Stain/Dye||Residues associated||Sensitivity||Linear dynamic range||MS compatibility|
|Coomassie Brilliant Blue||Arginine Lysine||Good||1 order||Good|
|Sypro Ruby||Primary Amines||Excellent||3-4 orders||Good|
|Deep Purple||Primary Amines||Excellent||4 orders||Good|
|Minimal Cy Dyes||Lysine||Excellent||4-5 orders||Challenging|