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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