Standard Operating Procedure GARNet 8
Analysis of Arabidopsis thaliana for Polyamines.
Author: Jennie Lewis
Last Modified: February 2003
Standard Operating Procedure:
Analysing Arabidopsis thaliana plants for Polyamines.
Materials:
Equipment:
1.5ml Eppendorf tubes
Percholric acid
1,6 Diaminohexane
Sodium carbonate
Dansyl Chloride
L-Proline
Acetone
Polished Water
Toluene
Acetonitrile
Glass vials
Injection vials and caps
Tapered low volume inserts
Gilson pipettes and tips
Balance
Whirlimix
Centrifuge
Oven
Glass syringes
HPLC with Diode Array Detector
Column: ODS-5 5μ 250x4.6mm
Guard Cartridge: ODS-5 5μ
Procedure:
Step 1: Plant Extraction
1.1
Make up the following stock solutions:
 0.2M Perchloric acid (100ml)
 0.01M 1,6 Diaminohexane (100ml)
 Saturated Sodium Carbonate (20ml)
 Dansyl Chloride 10mg/ml in acteone (3ml)
 L-Proline 100mg/ml in water (1ml)
Store all the solutions at 4-8ºC
1.2
Label three 1.5ml eppendorf tubes with the plant material to be
analysed and Replicate 1, 2 or 3.
1.3
Weigh out 100mg fresh, frozen plant material, prepared for analysis
following SOP GARnet 4, into each labelled eppendorf tube.
1.4
To each tube add 1ml chilled 0.2M Perchloric acid and 10μl of the
internal standard 0.01M 1,6 Diaminohexane.
1.5
Mix the plant material and solvent by holding the tubes in a bench top
whirlimix for 1 minute.
1.6
Leave the tubes to stand on the bench for 4 minutes
1.7
Repeat steps 1.5 and 1.6 and then 1.5 once more.
1.8
Centrifuge the tubes for 5 minutes.
Page 1 of 3
Standard Operating Procedure GARNet 8
Analysis of Arabidopsis thaliana for Polyamines.
Author: Jennie Lewis
Last Modified: February 2003
Step 2. Dansylation
2.1
Label glass screw top vials with plant material and replicate number.
2.2
Transfer 100μl of supernatant into the correctly labelled vial.
2.3
Add 200μl saturated sodium carbonate to each vial.
2.4
Add 400μl dansyl chloride (10mg/ml in acetone) to each vial.
2.5
Mix each vial for 10 seconds using a bench top whirlimix.
2.6
Heat the vials in the dark at 60ºC for 1 hour.
2.7
Remove the vials from the oven and add 100μl L-Proline (100mg/ml in
water).
2.8
Return the vials to the oven and heat at 60ºC in the dark for a further ½
an hour.
2.9
Remove the vials from the oven and with a glass syringe add 500μl
Toluene.
2.10
Mix each vial for 30 seconds using a bench top whirlimix. Leave to
stand on the bench until clear partitioning can be seen.
2.11
Label glass screw top vials with plant material and replicate number
and aqueous phase.
2.12
Transfer the bottom aqueous layer, with a glass syringe, to the
appropriately labelled glass vial.
2.13
Dry each top phase with Nitrogen.
2.14
With a glass syringe add 200μl acetonitrile to each dry sample.
2.15
Mix each vial for 30 seconds using a bench top whirlimix and then
leave the vials to stand on the bench for 30 minutes.
2.16
Label injection vials with the appropriate plant material and replicate
number and into each place a tapered low volume insert.
2.17
After 30 minutes transfer 60μl of each sample, with a glass syringe, to
the labelled injection vial and cap the vial.
Page 2 of 3
Standard Operating Procedure GARNet 8
Analysis of Arabidopsis thaliana for Polyamines.
Author: Jennie Lewis
Last Modified: February 2003
2.18
Store the remaining supernatant, the aqueous layer and the remaining
sample (in Acetonitrile) in labelled glass vials at 20ºC.
Step 3: HPLC Analysis
2.1
Make up Mobile Phases A: Water and B: Acetonitrile.
2.2
Set up the HPLC with a ODS-5 5μ, 250x4.6mm column with in line
guard cartridge.
2.3
Programme the HPLC with the following gradient and flow rate of
1ml/min.
Time (minutes)
0
30
35
40
45
50
% Mobile Phase A
40
10
0
100
40
40
% Mobile Phase B
60
90
100
0
60
60
2.4
Set the diode array detector to scan at 250-550nm and to selectively
monitor at 365nm and 425nm.
2.5
Equilibrate the column in 40% Mobile Phase A until a flat base line is
maintained.
2.6
Inject 25μl of each prepared sample in to the HPLC system.
Page 3 of 3