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
||Linear dynamic range
|Coomassie Brilliant Blue
|Minimal Cy Dyes