Developing a Western Blot Assay for 1-Aminocyclopropane-1-Carboxylic Acid in Tomatoes
Location
CSU Ballroom
Start Date
20-4-2015 10:00 AM
End Date
20-4-2015 11:30 AM
Student's Major
Chemistry and Geology
Student's College
Science, Engineering and Technology
Mentor's Name
James Rife
Mentor's Email Address
james.rife@mnsu.edu
Mentor's Department
Chemistry and Geology
Mentor's College
Science, Engineering and Technology
Description
Ethylene is a plant hormone involved in up-regulating plant defense responses and signaling fruit maturation. The final step in ethylene biosynthesis is the conversion of 1-aminocyclopropane-1- carboxylic acid (ACC) to ethylene by ACC oxidase (ACO). Previous studies have shown that methyl jasmonate (MeJ) treatment and tissue wounding increases ethylene production and up-regulates the transcription of ACO mRNAs. The focus of this study was to develop a Western blotting technique to probe ACO protein levels in tomato plant tissue and to use the assay to compare levels of ACO in MeJ treated and mechanically wounded plants relative to the control group. Plants were grown in laboratory under identical conditions. The plants were treated with MeJ, mechanically wounded with forceps, or left untreated (control). Samples of plant leaf tissue were then taken at 0, 3, 6, and 24 hours after the applied treatment. These samples were homogenized and the proteins extracted. Total protein extracts were then separated by SDS polyacrylamide gel electrophoresis and the bands were transferred to a nitrocellulose membrane. The membrane was then probed with ACC oxidase (aN- 19) antibody (Santa Cruz Biotechnologies) and with EIF4E1 antibody as an endogenous control Abiocode). The membrane was then probed with secondary anitbodies labeled with IR Fluorescent dyes (IRDye® 680LT labeled donkey anti-goat and IRDye® 800CW labeled donkey anti-rabbit). Finally, the proteins were visualized using the LI-COR Odyssey™ Imager and the resulting data was normalized against the endogenous control.
Developing a Western Blot Assay for 1-Aminocyclopropane-1-Carboxylic Acid in Tomatoes
CSU Ballroom
Ethylene is a plant hormone involved in up-regulating plant defense responses and signaling fruit maturation. The final step in ethylene biosynthesis is the conversion of 1-aminocyclopropane-1- carboxylic acid (ACC) to ethylene by ACC oxidase (ACO). Previous studies have shown that methyl jasmonate (MeJ) treatment and tissue wounding increases ethylene production and up-regulates the transcription of ACO mRNAs. The focus of this study was to develop a Western blotting technique to probe ACO protein levels in tomato plant tissue and to use the assay to compare levels of ACO in MeJ treated and mechanically wounded plants relative to the control group. Plants were grown in laboratory under identical conditions. The plants were treated with MeJ, mechanically wounded with forceps, or left untreated (control). Samples of plant leaf tissue were then taken at 0, 3, 6, and 24 hours after the applied treatment. These samples were homogenized and the proteins extracted. Total protein extracts were then separated by SDS polyacrylamide gel electrophoresis and the bands were transferred to a nitrocellulose membrane. The membrane was then probed with ACC oxidase (aN- 19) antibody (Santa Cruz Biotechnologies) and with EIF4E1 antibody as an endogenous control Abiocode). The membrane was then probed with secondary anitbodies labeled with IR Fluorescent dyes (IRDye® 680LT labeled donkey anti-goat and IRDye® 800CW labeled donkey anti-rabbit). Finally, the proteins were visualized using the LI-COR Odyssey™ Imager and the resulting data was normalized against the endogenous control.
Recommended Citation
Close, David. "Developing a Western Blot Assay for 1-Aminocyclopropane-1-Carboxylic Acid in Tomatoes." Undergraduate Research Symposium, Mankato, MN, April 20, 2015.
https://cornerstone.lib.mnsu.edu/urs/2015/poster_session_A/36
