High-resolution 2-Dimensional Electrophoresis (2DE) gels can be run either in vertical or horizontal gel systems. The Aberdeen Proteome Facility (APF) uses vertical systems throughout. The following notes provide a brief guide on the selection of the 2D gel format and subsequent processing for your analyses and will aid in the planning of your work.
Carrier ampholytes are the traditional method for the first dimension separation of 2DE. With suitable care CAs can provide excellent data but there are many problems in their use. In contrast Immobilised pH Gradients (IPGs) have many advantages not least their reproducibility between runs. IPGs can be used as either broad and narrow range pH gradients and they are less sensitive to sample contaminants compared to CA separations. The APF use IPGs purchased from Amersham-Pharmacia Biotech. Although IPGs are more expensive than gels prepared with CAs this extra cost is outweighed by their advantages.
Small format gels are both cheap and rapid. They are ideal for pilot studies and for the analysis of low complexity samples. With the use of IPG gels they can provide perfectly usable data for many projects. Proteins can be successfully cut from small format gels for peptide mass mapping. Large format gels can provide improved resolution and allow higher protein loads (helps to detect the low abundance proteins in a sample). However, you need larger amounts of sample compared to the equivalent small format gel. Large format gels are more expensive and do take longer to process compared to small format gels.
Two methods are used for the post-electrophoretic detection of proteins. Colloidal Coomassie Brilliant Blue G250 staining has a low sensitivity but has advantages of providing good quantitative data and is also compatible with subsequent peptide mass mapping. Silver staining has much improved (ca. 10-fold) sensitivity for detection but is not ideal for quantitation for a variety of reasons. In addition, silver staining is not compatible with the current methods of peptide mass mapping used at the APF - this may change in the future.
Visual inspection of 2D protein profiles is quick and easy and can be achieved by simply overlaying the dried gels. If you know what you are looking for (i.e. which spot(s)) and are not interested in the other protein spots then manual comparison may be the easiest option. Computer analysis is very time-consuming and can, if not done carefully, lead to many subjective decisions and errors in spot identification. However, when done correctly computer analysis can provide high quality quantitative data for all spots in the 2D profile. Computer analysis is only worth doing if your are analysing high quality 2D profiles.
You may be able to process a single gel for each sample if you are carrying out a pilot study. However, always be prepared to do repeat analyses to confirm the data. If you are doing quantitation of protein synthesis by computer analysis then samples will need to be processed in quadruplicate or better. It is for you to decide if replicates means replicate gels of the same sample or replicate samples run on each gel.
The main point to stress is that you should plan the investigations in advance and decide what you want to achieve before starting processing samples by 2DE. You can always contact the lab to discuss your plans - there are certain to be options not covered in the above.
Phil Cash
Telephone: 01224-552505
Email: p.cash@abdn.ac.uk
Evelyn Argo
Telephone: 01224-552505
Email: e.argo@abdn.ac.uk
Audrey Innes
Telephone: 01224-552505
Email: i.innes@abdn.ac.uk
Web Sites
www.abdn.ac.uk/proteome
www.abdn.ac.uk/~mmb023/2dhome.htm
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