I want to determine ABA (abscisic acid) in citrus leaves...and need easy and implementable protocol for this purpose
Dear Hamza Sohail
You can quantify ABA content of leaves in two ways: 1) using gas chromatography–tandem mass spectrometry (GC-MS) and 2) application of immunoassays. The GC-MS method needs special facilities, but the antibody based method is relatively simple and can be carried out in laboratory.
Procedure
Collect fresh leaves and freeze leaf tissues (200 mg) with liquid nitrogen immediately.
Carefully grind them in a mortar and pestle to get the fine powder.
Add 500 µl extraction buffer to mix them thoroughly by gently pipetting.
Transfer extracts to a covered, siliconized borosilicate tube.
Incubate the extracts overnight in darkness at 4 °C.
Centrifuge at 8, 000 x g for 10 min at 4 °C.
Transfer supernatant to pre-cold new 1.5 ml Eppendorf tube.
Vacuum centrifuge at 4 °C to evaporate the supernatant.
Dissolve the residue with 500 µl methanolic Tris buffer (if necessary, gently pipette up and down a few times).
Measure the ABA content with Phytodtek ELISA kit according the manufacturer’s instructions.
· Extraction buffer
90% (v/v) methanol
200 mg/L sodium diethyldithiocarbamate trihydrate
· Methanolic tris buffer
10% methanol
50 mM Tris (pH 8.0)
1 mM MgCl2
150 mM NaCl
Dear Hamza Sohail
Pls. find the attached files
http://jxb.oxfordjournals.org/content/early/2014/04/03/jxb.eru148.full
http://www.nature.com/articles/srep20212
http://link.springer.com/article/10.1007/s11738-010-0706-4
http://www.crec.ifas.ufl.edu/academics/classes/hos6546/materials/scientia_horticulturae_42_257-267.pdf
http://www.publish.csiro.au/fp/FP11269
https://www.ncbi.nlm.nih.gov/pubmed/22888124
Martínez-Cortina C, Sanz A (1993) Effect of hormone on sucrose uptake and on ATPase activity of Citrus sinensis L. Osbeck leaves. Ann Bot 73:331–335
http://link.springer.com/article/10.1007/s11738-012-0947-5
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431003/
Hoping this will be helpful
Regards
Prof. Houda Kawas
we use the following for the extraction and determination of ABA : 1.Extract fresh leave samples using 80% methanol for 48 hours at 4C in darkness, during which time ,methanol is changed twice. 2. combine the methanolic extract and evaporate at 45C to aqueous phase,using cooled rotary evaporator.3. make the the aqueous phase to 50ml with distilled water. make the PH 2.5 with dilute HCl. then do the process of partitioning using separator funnel and collect the ether phase.4.Evaporate the ether phase to 1ml. then, ABA can be determined using High Pressure Liquid Chromatography,using C-18 reversed phase column, packed with 5 micrometer ODS,. The mobile phase is 0.1M phosphoric acid:methanol 60:40v/v at PH2.5. the flow rate at 1.0 ml per minute . standared ABA pared at different concentration in should be prepared in phosphoric acid methanol . detection is done by uv absorption at 254nm. i
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