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).
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
pH to 5 with acetic acid
SA standard: 1 mM
- Grind your tissue in liquid nitrogen and weight out between 0.1g and 0.5g of leaf tissue and place in 2 ml tubes
- 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)
- Spin sample for 20 minutes at full speed on a bench top centrifuge
- 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
- Combine this new sup with sup from step 4, mix well.
- 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).
- Split all samples in two equal parts, one half will be used for total SA quantification the other half for free SA.
- Dry in the hood overnight or dry in a speedvac until you have between 10-50 mL left.
- 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.
- For free SA determination add 0.5 mL TCA to sample vortex and sonicate for 5 minutes.
- Spin samples for 15 minutes, transfer to a new tube.
- 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.
- Dry sample in speed-vac (approx 25 minutes)
- Resuspend in 250 mL mobile phase vortex and sonicate for 5 minutes.
- Spin @max speed 5 minutes and transfer to a new tube (to remove any residue)
- Proceed with HPLC or store @-20C.