Plant tissue protein extraction

They also suggested the lipids were released and became extractable primarily through enzymatic reactions after maceration of the leaves for extraction. Thus, they postulated that the time interval between maceration of the plant tissue and the application of heat to coagulate proteins was critical. They recommended heat inactivation of enzymes and precipitation of proteins as quickly as possible after maceration to minimize this interference. 

Preparation of Other Antigenic Materials. Cotton plant tissues (stem, leaf, burr), cotton gin trash, baled cotton, clean cotton lint, both hand picked in the field and from plants grown in the greenhouse, cottonseed proteins, cottonseed hulls, house dust, and flax, soft hemp, sisal, and jute fibers, were extracted with deionized water. The purification process was, however, stopped to correspond to f-3 (see Figure 1). 

Automated robotic extraction of proteins from plant tissues... 

When plant or animal tissues are extracted with nonpolar solvents, a portion of the material dissolves. The components of this soluble fraction are called lipids and include fatty acids, triacylglycerols, waxes, terpenes, postagladins, and steroids. The insoluble portion contains the more polar plant components including carbohydrates, lignin, proteins, and nucleic acids. 

The extraction and purification of proteins from organisms or biological tissue can be a laborious and expensive process, and often represents the principal reason why vaccines and other therapeutic agents reach costs that become unattainable for many. Downstream processing also can be a major obstacle with respect to cost for large-scale protein manufacturing in plants. However, purification from plant tissues, while still costly, is in general less expensive than purification from their mammalian and bacterial counterparts. Indeed, some plant-derived biopharmaceuticals, such as topically applied monoclonal antibodies, may require only partial purification and thus be even less intensive in terms of labor and cost. 

Protein fractions having lectin activity have been extracted from mitochondria, plasma, and Golgi membranes, and from the endoplasmic reticulum of mung-bean hypocotyls, as well as from total-membrane fractions from a variety of plant tissues.257 The carbohydrate specificity of the lectin fractions differs with the membrane type. It is conceivable that, in addition to cell-wall lectin-membrane interactions, there may also be membrane lectin-cell wall, noncovalent bonds. 

Common technical approaches to plant proteomics investigations are the extraction and the purification of proteins from crude tissue (8) however, due to the presence of a number of proteases and a low protein charge, a protection against proteolysis is highly recommended by adding selected inhibitors. Then the removal of nonproteinaceous products is operated according to the nature of the plant tissue (see scheme on Figure 2). 

Plant tissue collection for DNA and protein studies has primarily utilized leaf tissue, but seeds, roots, flowers, stems, pollen, spores, and game-tophytes have all been used successfully. For example, DNA extraction from the parasitic Cuscuta required using only intemode tissue to prevent DNA contamination from its host species26 likewise, the green stems of a leafless Koeberlinia provided an adequate source of DNA.27... 

The second approach to storage of plant tissue is to minimize or even eliminate storage in ultracold fieezers and to store only the DNA permanently. Plant tissue is placed in the refrigerator as it arrives, and then DNA extraction is done as soon as possible. This is the preferred method (e.g., J. Palmer laboratory)11 in studies where fast DNA extraction methods work well, tissue can be processed quickly, and no backup tissue source for additional extractions of either DNA or proteins is needed or expected. The benefits of this approach include the following (1) the need is diminished for ultracold storage space, which is costly in terms of purchasing, electrical power, and maintenance (2) space is then available for more... 

As is the case with other biotechnological products, the extent of protein purification depends on the final intended application of the product. There are applications where the plant tissue can be directly used, and hence purification is not needed. In other situations particularly pharmaceutical products administered parenterally, there are stringent purity requirements, necessitating complete removal of viral particles, endotoxins and other contaminants. There are very few published reports that make quantitative and characterize the extraction and purification of proteins from transgenic plants. Furthermore, there are practically none dealing with the economics of their downstream processing. 

Isoelectric focusing and electrophoresis were used for protein mapping and to study protein extraction in horizontal ultra-thin-layer format [138], The 0.12-0.36-mm-thick polyacrylamide gel layer was deposited onto tiny glass plates (e.g., microscope slides). The method enabled the analysis of 1-ng tissue culture specimens. Ultra-thin-layer polyacrylamide isoelectric focusing gel was also employed in two-dimensional analysis of plant and fungal proteins. Marlow et al. [139] reported on the use of 0.2-mm semirigid backing (polyester)-supported... 

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