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.