By Reiner Westermeier
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Additional resources for Electrophoresis in practice: a guide to methods and applications of DNA and protein separations
An alternative way to adequate and reproducible 2-D pattern in basic gradients is suggested by Hoving et al. (2001): the addition of higher amounts of DTT to the gel, adding more DTT to a cathodal paper strip, and a few more measures described later in the isoelectric focusing chapter. Hoving S, Gerrits B, Voshol H, Müller D, Roberts RC, van Oostrum J. Proteomics 2 (2002) 127±134. Carrier ampholytes Carrier ampholytes, which had been designed for generating pH gradients, improve the solubility of proteins considerably by substituting ionic buffers.
Sometimes lyophilized cells are brought into solution with lysis solution. For analytical applications 5 ± 10 106 cell equivalents are usually applied. Bacteria cells can be extracted by repeated freezing at ±20 C and thawing. For some organisms detergent lysis is necessary, for instance for tough cell walls of yeast and fungi. 1 % (w/v) SDS prior to IEF. Sonication with a probe is very helpful for solubilisation. French pressure cells and mechanical homogenizors are employed to burst tough cell walls from yeast or plant cells.
Plant proteins Sample preparation from plant tissues is particularly troublesome, because interfering substances such as polysaccharides, nucleic acids, lipids and phenolic compounds are present in high concentrations, whereas the proteins exist in low abundance. Usually the proteins have to be precipitated as described above on page 21. More complicated procedures are required for the analysis of plant membrane proteins (Hurkman and Tanaka, 1986). The protein pellet has to be extensively washed before it can be resuspended in lysis buffer for IEF.