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Sample Preparation for 2D Analysis
Sample Preparation
Samples should be supplied free of the following components that interfere with the first dimension isoelectric focusing step: salt, nucleic acids, polysaccharides, and lipids. Salt is a particular problem, as it leads to burning of IPGH strips. We recommend the following desalting methods:- Methanol
precipitation
- TCA/acetone precipitation
- Spin columns e.g. Centricon
Amount of Material
The following is a table giving a guideline to the amounts of protein required for various gel types: Analytical load (silver) mini gel 10-50ug total proteinAnalytical load (silver) large gel 100-200ug total protein
Preparative load (coomassie) mini gel 100-500ug total protein
Preparative load (coomassie) large gel 1-3mg total proteinIf you are unsure of protein concentrations and need to know how much sample to send, the following is an approximate guide:
- Bacteria are approx. half their dry-weight in protein
- Plasma/Serum is approx. 60mg/ml protein
- 1x107 Mammalian cells is approximately equal to 1mg of
protein
- Mammalian tissue (e.g. rat liver) requires a minimum
of 10mg freeze dried material
- Human Urine anywhere between 0.5mL to 10mL needed for
a large analytical gel ("normal" control people will
require 10mL)