Download Acidic Plant Hormone Analysis - Donald Danforth Plant Science

Acidic Plant Hormone Analysis - Customer Instructions
Scope:
Compounds quantified: Jasmonic acid (JA), Salicyclic acid (SA), Abscisic acid (ABA), Indole-3-acetic acid (IAA), JA-Ile
(jasmonoyl-isoleucine), OPDA (12-oxo-phytodienoic acid), IAA-Asp (Indole-3-acetyl-aspartic acid).
Biological materials: The method is suitable for most vegetative plant tissues, including leaves, roots, fruits and seeds. It
is recommended that you provide a few extra samples together with your batch samples for testing the grinding
procedure, extraction, and matrix effects.
Experimental design
•
Experimental design is the responsibility of the researcher. However, please feel free to contact the facility if you
would like advice or help in designing your experiment. Below are some guidelines to help minimize biological
and experimental variation:
™ Replication reduces variability in experimental results. We suggest analyzing at least 3 biological
replicates per sample or treatment to reduce the variability, and increase the confidence level of each
treatment
™ A replicate sample should be a pool of several samples (ex: one biological replicate within a treatment is
derived from harvesting and pooling several leaves from several plants and not just one). Experiments
were performed internally to test the biological variation between samples coming from within the same
plant and between individual plants. Results showed that there is a high variation if replicates are not a
pool of tissue from several plants
Note:
•
•
In the case of IAA detection, for many plant samples the matrix of the extracted tissue is complex and leads to
high background and ion suppression (esp. leaf and seed samples). We have available a, more sensitive method
to detect IAA, at an additional cost. In this case a 40 min LC-MS run is required. Please specify if you wish to
request this additional analysis.
IAA-Asp will be reported as a relative amount (peak area only) because there is no stable isotopically-labeled
internal standard available for absolute quantitation.
Specification of this protocol (linearity, LOD, LOQ of the standards)
Dilution
SA
JA
IAA
ABA
IAA-Asp
JA-Ile
OPDA
Series unit
(μM)
Diln-8 0.000128
0.000128
0.000128
0.000128
0.000128
0.000128
0.000064
0.000064
Diln-7 0.00064
0.00064
0.00064
0.00064
0.00064
0.00064
0.00032
0.00032
Diln-6 0.0032
0.0032
0.0032
0.0032
0.0032
0.0032
0.0016
0.0016
Diln-5 0.016
0.016
0.016
0.016
0.016
0.016
0.008
0.008
Diln-4 0.08
0.08
0.08
0.08
0.08
0.08
0.04
0.04
Diln-3 0.4
0.4
0.4
0.4
0.4
0.4
0.2
0.2
Diln-2 2
2
2
2
2
2
1
1
Diln-1 10
10
10
10
10
10
5
5
• 5x serial dilutions of unlabeled hormone standards
• Areas darkened are outside of the linear range. Concentrations in white are the ranges that will be quantified
using this protocol
Procedure
Please follow the instructions below to ensure prompt analysis of your samples
Preparation of samples
•
Weigh out the appropriate amount of tissue for each sample, and send the information electronically (Excel file).
Additionally send a hard copy of the list of samples submitted with the samples. 100-150 mg of plant tissue (FW)
is sufficient for analysis in most cases. Additional sample preparation time will be charged to the customer if
facility staff needs to weigh out submitted samples.
•
Please provide your samples in 2mL microfuge tubes (preferably with snap-lock top; ex Eppendorf Tube cat # C3247-1). Make sure your sample tubes are clearly labeled.
•
Freeze samples as quickly as possible after harvesting, and store at -80ºC prior to submission. Send frozen
samples with sufficient dry ice, to ensure they remain frozen in transit to the facility.
•
Lyophilized plant material is also suitable for analysis. In this case, only 50-70mg tissue is required.
•
For internal (Danforth) clients, we can provide you with appropriate tubes for your samples. Please request these
in advance of preparing your samples.
Publication Acknowledgement:
•
If any results from this method are being used for publication or grants, it is greatly appreciated to acknowledge
the Proteomics & Mass Spectrometry Facility at the Danforth Plant Science Center for its contribution. Additionally
a write up of the hormone methods is available upon request.
External Fees:
Academic
• Sample Preparation - $80/hour, charged based on time required
• LC-MS/MS - $60/sample
• LC-MS/MS (high sensitivity for IAA) - $90/sample
• Data Analysis - $80/hour, charged based on time required
Commercial
• Sample Preparation - $95/hour, charged based on time required
• LC-MS/MS - $75/sample
• LC-MS/MS (high sensitivity for IAA) - $130/sample
• Data Analysis - $90/hour, charged based on time required