 |
||
 |
 |
 |
 |
||
 The CNRU Mass Spectrometry Core offers Proteomics and Metabolomics workshops.
|
|
|
|||||||||||
 |
 |
||||||||||
SAMPLE FRACTIONATION AND PREPARATIONDue to limits in loading capacity, some simplification and cleanup of complex biological samples is almost always necessary to successfully resolve proteins using two-dimensional gel electrophoresis. An understanding of your biological system of interest and your goals will help the staff at the CNRU Mass Spectrometry Core Facility to design an analytical strategy suitable for your sample. For instance, you may be interested in proteins present in an organelle or bound to cell membranes. You may specifically be interested in nuclear or cytoplasmic proteins. Specific sample fractionation will simplify your mixture and produce better results. Most of the commercially available kits for fractionation will leave your fractionated sample in a 2D-compatible buffer.The goal of sample fractionation is to simplify your mixture of proteins. There is a limit to how much material can be loaded onto a two-dimensional gel, as well as a limit on how many proteins can be resolved in each dimension. Fractionating your sample will allow you to load more of the proteins of interest onto a gel, will increase the probability of separating and resolving proteins so only one protein will migrate to a given spot, and will improve the quality of your mass spectral analyses subsequent to the gel for better protein identification. At the most basic level, the final goal of sample preparation is to completely denature and solubilize your fractionated proteins in a buffer that is compatible with 2DGE. There are many ways to achieve this goal and the method you choose will be dependent on your objectives and the fractionation methods you use prior to 2DGE. The final sample buffer can vary somewhat in its components but always includes Urea (8-9M), a reductant (usually DTT) and one or more detergents. Click for general information on developing a workflow strategy using 2DGE. Limitations on loading also require that your sample quantity is properly measured. Sample cleanup and concentration may be required before and after quantitation. PROTOCOLSPlease follow the links to basic protocols that can be adapted to suit your sample preparation needs. Please note that these are intended for preliminary experiments only; optimum conditions will need to be determined empirically for individual samples.
An excellent resource is available in a booklet from Amersham entitled "2D Electrophoresis using immoblilized pH gradients. Principles and Methods". You may also find useful information in the booklet from BioRad "2D Electrophoresis for Proteomics". |
|||||||||||
|
|
 |
|||||||||
