To investigate novel pathways involved in auxin biosynthesis, transport, metabolism, and response, we have developed a high-throughput screen for indole-3-acetic acid (IAA) levels. Historically, the quantitative analysis of IAA has been a cumbersome and time-consuming process that does not lend itself to the screening of large numbers of samples. The method described here can be performed with or without an automated liquid handler and involves purification solely by solid-phase extraction in a 96-well format, allowing the analysis of up to 96 samples per day. In preparation for quantitative analysis by selected ion monitoring-gas chromatography-mass spectrometry, the carboxylic acid moiety of IAA is derivatized by methylation. The derivatization of the IAA described here was also done in a 96-well format in which up to 96 samples can be methylated at once, minimizing the handling of the toxic reagent, diazomethane. To this end, we have designed a custom diazomethane generator that can safely withstand high flow and accommodate larger volumes. The method for IAA analysis is robust and accurate over a range of plant tissue weights and can be used to screen for and quantify other indolic auxins and compounds including indole-3-butyric acid, 4-chloro-indole-3-acetic acid, and indole-3-propionic acid.
Bibliographical noteFunding Information:
This work was supported by NSF DBI 0077769 to J.D.C. and J.N., and in part by USDA-NRI 2005-35318-16197 to J.D.C. and USDA-NRI 2002-03555 to J.N. We are grateful to Camille Jensen, Vikesh Patel, and Yazmin Mohammad for conducting many trials of the extraction protocols. We thank the many visitors to our laboratories over the past 5 years who tested our procedures as they were developed. We also thank Dan Bensen for the design and production of the custom metal bead dropper, insert plate, and diazomethane generator.
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- 4-Chloro-indole-3-acetic acid
- High-throughput methylation
- High-throughput purification
- Indole-3-acetic acid
- Indole-3-butyric acid
- Indole-3-propanoic acid
- Organic acids
- Plant hormones