TY - JOUR
T1 - A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue
AU - Barkawi, Lana S.
AU - Tam, Yuen Yee
AU - Tillman, Julie A.
AU - Pederson, Ben
AU - Calio, Jessica
AU - Al-Amier, Hussein
AU - Emerick, Michael
AU - Normanly, Jennifer
AU - Cohen, Jerry D.
PY - 2008/1/15
Y1 - 2008/1/15
N2 - 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.
AB - 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.
KW - 4-Chloro-indole-3-acetic acid
KW - 4-Cl-IAA
KW - Auxin
KW - High-throughput methylation
KW - High-throughput purification
KW - IAA
KW - IBA
KW - IPA
KW - Indole-3-acetic acid
KW - Indole-3-butyric acid
KW - Indole-3-propanoic acid
KW - Organic acids
KW - Plant hormones
UR - http://www.scopus.com/inward/record.url?scp=36148952909&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=36148952909&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2007.08.009
DO - 10.1016/j.ab.2007.08.009
M3 - Article
C2 - 17889819
AN - SCOPUS:36148952909
VL - 372
SP - 177
EP - 188
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -