1. Soybean seedlings were germinated in the dark for 5-6 days. Note: the seeds should be germinated under
conditions where just enough water is added for the 5 day germination period. As a yard stick, add enough water to make sure that the paper towels are wet, leaving about an addition 10-15 ml in the pans.
2. Cut of roots and freeze in liquid nitrogen.
3. To extract inducer, thaw the root tissue, and add Ethanol (Optima grade; Fisher Scientific) in the following ratio: 2 ml Ethanol for every gram of tissue. Typically 100-150 g of tissue is used.
4. Grind tissue in waring blendor, and place mixture in clean glass flask (500 ml) and shake 2 h at RT. The mixture is left overnight in the cold room.
5. Spin mixture JA17 (40000 rpm) to remove root debris. Keep supernatant.
6. Rotary vap supernatant to concentrate 10 fold. Starting with 100 gram of tissue, this usually results in about
10-15 ml of concentrated SSG. Store at -20 C. Use about 5 ul of sample in assays with the nolA-lacZ fusion. Increasing the amount of SSG does not increase activity.
Isolation of Inducer
1. Prepare Sep-pak as per manufacturers instructions. This entails first washing the column sequentially with100% MeOH, and water. Add 2 ml of concentrated SSG to the sep-pak, reapplying flow through 3 times. Keep flow through.
2. The Sep-pak column is then washed with 3 x with water, 3 x with 60% MeOH and then eluted with 3 x 100% MeOH.
3. Eluted sample is concentrated by “air-drying” with the “house”-air. Prior to HPLC, the sample is resuspended in 5 ml 60% MeOH. Note: the sample is very concentrated at this juncture, and this fraction is usually diluted 4-5 fold (ie. 500 ul of sample into 5 ml 60% MeOH) and applied to the HPLC.
4. The column used is: C18, phenomenex Jupiter, 250 x 4.6 mm, 5 microns.
5. HPLC conditions: load sample 60% MeOH/40% water.
1 min: 60% MeOH
40 min 100% MeOH
hold 15 min at 100% MeOH
return (5 min) to 60% MeOH.
6. Active fractions are highlighted in the attached. Peak No. 4 is chitinase sensitive. Peaks,12, 12a and 13 are active on the nolA fusion but are not chitinase sensitive.
XAD column Protocol
1. As an alternative means to boost inducer isolation, the following protocol was utilized to enrich for inducer.
2. XAD column matrix was prepared as directed by the manufacturer. (Typically 5 g of matrix was used for each XAD based purification).
3. XAD beads were transferred to a 500 ml beaker and MeOH added to cover the resin. The beads were soaked for 15 min in MeOH.
4. Methanol was replaced, and the beads were washed with distilled water.
5. Washing with distilled water was repeated 3 times to remove all residual MeOH, and the beads allowed to sit in water for 10 min.
6. The slurry was then removed and added to 5 ml of SSG extract (the SSG extract can be diluted at this stage with water to make up a total volume of about 15 ml).
7. This SSG-XAD slurry was incubated overnight with gentle rocking at RT in the dark in 20 ml vials containing PFTE caps.
8. Following incubation, the suspension was allowed to sit and the unbound SSG removed. The beads were washed with water 5 times, and eluted first with 100% MeOH (4 times on a rotary shaker at 150 rpm). The MeOH fractions were pooled.
9. The remaining bound material was then eluted with acetone.
10. Both MeOH and acetone eluted fractions were air dried separately, resuspended separately in water before being applied to the sep-pak column as described above. Following sep-pak purification, the samples were analyzed by HPLC as previously described.