Optimization of protein extraction from orchids : [an honors thesis (HONRS 499)]

Abstract

There is a relatively small amount of information known about plants' responses to pathogens, and there is almost no information specifically concerning orchids' responses. One way to analyze the response of orchids to pathogens is to analyze protein expression using twodimensional SDS polyacrylamide gels. In this research project, protein extraction from orchids was optimized to yield pure, soluble protein samples. Protein extraction from plants is notoriously difficult, so two different protein extraction kits were used to try to extract a pure protein sample. One of the kits used was CelLytic-P (Sigma, St. Louis), which is specifically made for plant protein extraction. The other kit used was ProteoPrep (Sigma, St. Louis), which may be used on any type of cell or tissue, but which allows protein extraction from specific areas of the plant cells. Protein extraction using the ProteoPrep kit not only yielded different protein fractions from the plant cell, it also yielded significantly higher protein concentrations than the CelLytic-P kit. Therefore, the ProteoPrep kit will be used for protein extraction in further research. 2-D SDS PAGE electrophoresis was attempted two times using orchid tissue, but the gels showed only minor smearing. When running future gels, Silver stain will be used instead of less powerful Coomassie Blue stain. For future research, five orchid clones were inoculated with Tobacco Mosaic Virus-Orchid strain (MV-0), and one clone was mock inoculated. Tissue was harvested at different times following infection and is being stored for future protein extraction and analysis.Honors CollegeBall State UniversityMuncie, IN 47306