PROTOCOL 1.1 Isolation Glyoxysomes from Soybean Cotyledons
This protocol should be used for isolation of glyoxysomes from soybean cotyledons. We usually
use soybean seeds (Glycine max cv. Bansei Shirodaizu). Figure 1 shows the steps to isolate
glyoxysomes by Percoll density gradient centrifugation. Glyoxysomes are isolated from etiolated
soybean cotyledons germinated at 22 ºC in the dark for 7 days. All procedures should be carriedout at 4 ºC.
<Equipments and reagents>• Grinding buffer [20 mM pyrophosphate-HCl, pH 7.5, 1 mM EDTA, 0.3M mannitol]• Isolation buffer [10 mM HEPES-KOH, pH7.2, 1 mM EDTM, 0.3 M mannitol]• 50% (v/v) Percoll solutions [50% (v/v) Percoll, 10 mM HEPES-KOH, pH 7.2, 1mM
EDTA, 0.3 M raffinose]• 28% (v/v) Percoll solutions [28% (v/v) Percoll, 10 mM HEPES-KOH, pH 7.2, 1mM
EDTA, 0.3 M raffinose]• Homogenizer (SMT COMPANY, Japan)• Fractionator (SJ-1211; ATTO, Japan)• Centrifuge and centrifuge tubes

1. Prepare gradients that consist of 5 ml of 50% (v/v) and 30 ml of 28% (v/v) Percoll
solutions.
2. Harvest the soybean cotyledons (100 g fresh weight), and homogenize with 200 ml ofgrinding buffer in a chilled homogenizer twice for 3 seconds.
3. Squeeze the homogenate through four layers of cheesecloth, and homogenize theresidue with another 200 ml of grinding buffer.
4. Combine the filtrates and centrifuge at 1,500×g for 10 min to remove plastids and celldebris.
5. Centrifuge the supernatant at 10,000×g for 20 min, resuspend the pellet in 150 ml ofgrinding buffer, and centrifuge at 1,500×g for 10 min to remove remains of plastids andcell debris.
6. Centrifuge the supernatant at 10,000×g for 20 min, and then resuspend the pellet in 4 mlof isolation buffer.
7. Put the suspended solution on the top of the Percoll solutions, and centrifuge at
40,000×g for 40 min with slow acceleration and deceleration.
8. After centrifugation, fractionate samples into each 1.0 ml by the fractionator.
9. Measure catalase activity as a maker of glyoxysomes in each fraction.

PROTOCOL 1.2 Purification of Glyoxysomes
Figure 2 shows the steps to purify glyoxysomes by iodixanol density gradient centrifugation. Allprocedures should be carried out at 4 ºC.
<Equipments and reagents>• Isolation buffer [10 mM HEPES-KOH, pH7.2, 1 mM EDTM, 0.3 M mannitol]• 50% (w/v) Iodixanol solution [50% (w/v) Iodixanol, 25 mM sucrose, 2.5 mM
MOPS-HCl pH 7.2, 0.5 mM EDTA, 0.05% ethanol]• 15.5% (w/v) Iodixanol solution [15.5% (w/v) Iodixanol, 440 mM sucrose, 6 mM
MOPS-HCl pH 7.2, 1.2 mM EDTA, 0.12% ethanol]• 36% (w/v) Iodixanol solution [36% (w/v) Iodixanol, 170 mM sucrose, 4 mM
MOPS-HCl pH 7.2, 0.8 mM EDTA, 0.08% ethanol]• Ultracentrifuge, swinging rotor (SW 28.1; Beckman Coulter, San Diego, CA, USA), andtubes• Centrifuge and centrifuge tubes

1. Prepare gradients that consist of 2 ml of 50% (w/v) iodixanol solution and 13.6 ml of alinear iodixanol density gradient from 36% (w/v) to 15.5% solution. The gradientsshould be kept in 4 ºC for O/N.
2. Combine glyoxysomal fractions obtained from Percoll gradients, and dilute 5-fold withisolation buffer, followed by centrifugation at 4,800×g for 10 min.
3. Resuspend the pellet in 2 ml of isolation buffer.
4. Put the solution carefully onto the iodixanol density gradient.
5. Centrifuge at 100,000×g for 2.5 h in the SW28.1 swinging rotor.
6. Collect glyoxysomal fractions obtained from the iodixanol density gradient.

The fractions should be analyzed by immunoblotting to confirm that the peroxisomal fraction is
not contaminated with proteins of other organelles.

Cotyledon
Homogenization
Filtration

Percoll peroxisomalsolution
15.5 - 36% Iodixanol

Crude solution
C.f. 1,500xg, 10 min

50% Iodixanol

Supernatant
C.f. 10,000xg, 20 min

C.f. 100,000xg, 2.5 hPellet
Resuspend
C.f. 1,500xg, 15 min

Mitochondria etc.

Supernatant
C.f. 10,000xg, 20 min
Pellet:Crude peroxisomes
Resuspend
Crude peroxisomal
solution
28% Percoll

50% Percoll
C.f. 41,400xg, 40 min

Mitochondria etc.

Percoll peroxisomal
solution

Figure 1. Isolation Glyoxysomes from Soybean CotyledonsPurified peroxisomes

Figure 2. Purification of Glyoxysomes