Cell wall purification from Arabidopsis tissues

CELL WALL PURIFICATION FROM ARABIDOPSIS TISSUES

Last Update: December 2006

Reference: Zablackis E, J Huang, B Muller, AG Darvill and P Albersheim (1995) Characterization of the cell-wall polysaccharides of Arabidopsis thaliana leaves Plant Physiology 107:1129-1138

PREPARE SOLUTIONS

1. 10 % SDS (1 L):

Mix 100 g of SDS (Sodium dodecyl sulfate), 900 mL of dH₂O, Heat to 68^oC, pH to 7.2 w/ cHCl, add dH₂O to 1 L

2. 1.5% SDS (500 mL):

Mix 75 mL of 10% SDS with 425 mL dH₂O

3. 0.5% SDS (500 mL):

Mix 25 mL of 10% SDS with 475 mL dH₂O

4. a-amylase

Sigma A-6255

5. 1M KPO₄, pH 7.0 (100 mL)

Mix 61.5 mL of 1M K₂HPO₄, pH 7.0 with 38.3 mL of 1M KH₂PO₄

6. 0.1 M KPO₄ (1 L):

Mix 100 mL 1M M KPO₄ pH 7.0 with 900 mL dH₂O

7. 0.5 M KPO₄ (1 L):

Mix 500 mL 1M M KPO₄ pH 7.0 with 500 mL dH₂O

8. Phenol:Acetic Acid: Water (2:1:1) (400 mL):

Mix 200 mL Phenol, 100 mL acetic acid, 100 mL water

PROCEDURE

1. Harvest tissues/plants and freeze in liquid nitrogen and store at -70^oC

2. Lyophilize overnight

3. Throughly grind in liquid nitrogen (~1-2 g)

4. Extract with 1.5% SDS (~10 mL), homogenize, centrifuge for 5 mins at 3,000 rpms

5. Extract pellet with 0.5% SDS (~10 mL) twice, >15 minutes each, centrifuge for 5 mins at 3,000 rpms

6. Extract pellet with 0.1 M KPO₄ (~10 mL), centrifuge for 5 mins at 3,000 rpms

7. Extract pellet with 0.5 M KPO₄ (~10 mL), centrifuge for 5 mins at 3,000 rpms

8. Wash pellet with 0.5 M KPO₄ (~10 mL), followed by water, centrifuge for 5 mins at 3,000 rpms

9. Extract pellet with Phenol:Acetic Acid: Water (2:1:1) (~10 mL), centrifuge for 5 mins at 3,000 rpms

10. wash pellet with water

11. Dry with acetone: vortex to mix and centrifuge again. Remove most of the acetone and letair dry. Repeat if necessary

12. Digest cell wall with a-amylase: use 50 units of alpha-amylase / 10 mg of cell wall material / mL of 0.1 M KPO₄

13. Incubate for ~24 hours at 30^oC in shacking water bath (may repeat to ensure removal of all the starch). Centrifuge at 8,000g for 20 mins

14. Wash pellet with 0.1 M KPO₄, followed by a wash with water

15. Dry again with acetone

16. Store at -70^oC (transport in parafilm-sealed tubes in a bag containing Drierite/silica gel)