ISOLATION OF SOYBEAN SEEDLING NolA INDUCER
SSG Preparation
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 with
100% 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.