I have been searching for protocol of extraction for free SA and JA specifically from potato plants but still unsuccessful. Though I have found extraction protocols from rice, tobacco etc but I dont know if I can use these protocols for potato also. Moreover I want to extract SA and JA from roots and leaves, Can I use same protocol for both tissues and does extraction method is specific for analytical technique e.g. HPLC, GC-MS??
Try to look in solanaceae in general for extraction protocols (e.g. tomatoes). Otherwise, I would use HPLC cause derivatization really sucks
We routinely analyze JA and SA in plant material either roots, shoots or even fruits. We have applied it to several and different plant matrices. Please, check Durgbanshi et al. (2005) J. Agric Food Chem. 53, 8437-8442. Do not hesitate to contact me if you have any doubts.
You can follow Dobrev & Kaminek (2002) J Chromatrography A 950, 21-29. The analysis is by HPLC/MS and the extraction and purification by solid-phase extraction. Good luck!
Dear Sir,
Please read the following paper in the attached file.
Salicylic acid (SA) extraction protocol
If using this protocol please cite:
Li X,Zhang Y,Clarke JD,Li Y,Dong X. Identification and cloning of a negative regulator of systemic acquired resistance, SNI1, through a screen for suppressors of npr1-1. Cell 98(3):329-39. (1999).
Materials:
90% Methanol (HPLC grade)
100% Methanol (HPLC grade)
5% TCA (trichloroacetic acid)
b-glucosidase solution (80units/ml in 0.1M sodium acetate, pH 5,2)
Extraction buffer: Ethylacetate/cyclopentane/isopropanol: 100/99/1
Mobile phase for HPLC :
0.2M Potassium acetate
0.5mM EDTA
pH to 5 with acetic acid
Filter
De-gas
SA standard: 1 mM
1. Grind your tissue in liquid nitrogen and weight out between 0.1g and 0.5g of leaf tissue and place in 2 ml tubes
2. Add 0.6ml of 90 % methanol, vortex thoroughly and sonicate in a sonicator bath 20 minutes. Spike in a recovery control of 1 microliter of 1 mg/ml (SA in water)
3. Spin sample for 20 minutes at full speed on a bench top centrifuge
4. Transfer supernatant to a new 1,5ml tube. Re-extract leaf pellet with 0.5ml 100% methanol. Vortex thoroughly and sonicate in a sonicator bath 20 minutes. Spin for 20 minutes
5. Combine this new sup with sup from step 4, mix well.
6. Prepare 1 new tube containing 600 mL of 90% methanol + 500 mL of 100%+ 1 mL of 1 mg/ml (SA in water)(recovery control).
7. Split all samples in two equal parts, one half will be used for total SA quantification the other half for free SA.
8. Dry in the hood overnight or dry in a speedvac until you have between 10-50 mL left.
9. For total SA determination add 0.1 ml b-glucosidase solution to the almost dry samples, vortex and sonicate for 5 minutes. Incubate at 37C for 90 minutes then add 0.4 mL 5% TCA.
10. For free SA determination add 0.5 mL TCA to sample vortex and sonicate for 5 minutes.
11. Spin samples for 15 minutes, transfer to a new tube.
12. Extract 3 times with 0.5 mL extraction buffer. Add buffer, vortex well, spin 1 minute, transfer the top phase in a new tube, be careful not to take the lower phase.
13. Dry sample in speed-vac (approx 25 minutes)
14. Resuspend in 250 mL mobile phase vortex and sonicate for 5 minutes.
15. Spin @max speed 5 minutes and transfer to a new tube (to remove any residue)
16. Proceed with HPLC or store @-20C.
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