Differences in the structure, physiology and biochemistry of plant tissues often do not allow (for the extraction of their polypeptides) the application of current or conventional animal buffers and/or maceration techniques. The more complex macro-molecular composition (and interaction) within plant tissues (e.g., phenolic, carbohydrate, and hydrocarbon compounds), and the mechanical strength afforded by the cell wall necessitate more stringent maceration and isolation methods in plant extract preparation to ensure the integrity of the polypeptides.
Current plant polypeptide extraction methods favor the preparation of subcellular (component) enriched fractions of various tissues. Few methods are available which allow extraction of whole plant tissue polypeptides. In contrast, a number of more or less "universal" preparative techniques have been described for animal systems. In our hands, such methods proved to be unsuitable for the preparation of plant extracts. These methods are characterized by moderate ionic strength buffers, low detergent levels, disruption of tissue by osmotic shock with minimal mechanical maceration, and preparation at temperatures above 0 C.
We report below an improved method for the extraction of whole plant tissue which utilizes a modified buffer, a more rigorous protocol for tissue homogenization, and the use of polyacrylamide gradient pore gel electrophoresis.
Our methods evolved from an animal preparative technique outlined by
Atkinson (Dept. of Zoology, Univ. of Western Ontario). The components of
the animal buffer and the improved plant extraction buffer are contrasted
|Animal Extraction Buffer (AEB)||Plant Extraction Buffer (PEB)|
|80 mM Tris-HCl||200 mM Tris-HCl|
|2% SDS||5% SDS|
|5% 2-mercaptoethanol||7.5% 2-mercaptoethanol|
|20% glycerol||10 mM phenylmethylsulfonylfluoride (PMSF)|
|pH 6.8||pH 7.5|
Upon addition of buffer to the tissue, an extra mechanical grinding step employing a porcelain mortar and pestle was also found essential in obtaining a suitable yield of polypeptides from certain tissues (Baszczynski and Hughes, this Newsletter).
The increased PEB ionic strength has been found necessary in maintaining constant pH during extraction of plant polypeptides. Increased SDS and 2-mercaptoethanol concentrations resulted in heightened resolution of electropherograms (likely due to more complete denaturation/reduction of disulphide bonds and homogeneous detergent coating of protein subunits). Addition of the protease inhibitor PMSF to PEB decreased the amount of degradation products observed over time. The consistency of the tissue extract obtained with PEB made the addition of glycerol (used as an antioxidant and to increase the loading density of the samples) unnecessary. Use of a higher pH resulted in an increase in the number of polypeptides greater than 100 kilodaltons. The conditions described here were arrived at by the manipulation of each component individually until optimal results were obtained.
Polypeptides from maize pollen, primary root, shoot and mouse skeletal muscle tissues were extracted employing both PEB and AEB under identical conditions. These were separated simultaneously on 8.3% uniform concentration, 8.3-15% concave-exponential gradient and 3-15% linear gradient acrylamide gels in several replicates. In all cases the combination of more stringent maceration with PEB and use of gradient gels resulted in the heightened resolution of an increased number of bands. Differences in the relative amounts of polypeptides within tissues were noted.
These results are thought to be attributable to an increased polypeptide extraction efficiency or improved polypeptide solubilization, not achieved utilizing the corresponding animal method.
The conditions and methodology described in this contribution have allowed us to achieve improved polypeptide electrophoretic visualization from several tissues of such widely divergent species as corn, mouse and Neurospora crassa.
W. G. Hughes, C. L. Baszczynski and C. Ketola-Pirie
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