Improvements in the Protein Extraction Method of Disparate Tissues in Cotton.

2D electrophoresis is the process of separating and isolating the protein library of a species.  It is an incredibly powerful tool in identifying  minute changes that may be occurring within a tissue under a variety of conditions.  It has shown the ability to identify various protein changes that occur in cotton (Gossypium hirsutum) during environmental stressors.  However, a limitation of 2D electrophoresis is that following the 2nd dimensional run, the proteins are denatured.  If further examination on a particular tissue is required such as enzymatic characteristics, then a new sample of tissue is analyzed and relationships are extrapolated.  We have developed a simple extraction variation of both leaf and ovary homogenate that is both highly sensitive to antioxidant type analyses that can also be utilized for 2D electrophoresis.  Amounts of protein extracted from both tissue samples was significantly higher than protein concentrations that have been utilized in other practiced methods.  The homogenates also possessed an high conservation of antioxidants, such as glutathione reductase.  Our method uses protease inhibitors and polyphenolic binders to diminish oxidation and protease activities within the homogenates.  Results indicated more than a factor increase in the sensitivity of glutathione reductase measurements when compared to a previously used method in our labratory.  The same sample extractant also provided substantial amounts of protein to be utilized in further electrophoresis steps.