In vitro polysaccharide precipitation assays and fungal protection assays against chitinases

Polysaccharide Affinity Precipitation Assay

The polysaccharide precipitation assay is based on methods published in van den Burg et al (2006) and Tjoelker et al (2000).

• 30 mg of the polysaccharide substrate
• 15 µg of protein
• 500 µl of binding buffer (pH 5.5 buffer: 50 mM Sodium acetate, pH 5.5, 50 mM sodium chloride; pH7 buffer: 50mM NaCl, 50 mM Tris pH 7; pH 8 buffer: 50mM NaCl, 50 mM Tris pH 8)

1. Weigh 30 mg of the substrate into micro-centrifuge tube.
2. Add 500 µl volume of buffer, vortex well to mix, and make sure there are no dry clumps
3. Add the protein.
4. Incubate on shaker for 2 hours at RT, making sure substrate is mixing well with liquid.
5. Pellet at max rpm for 10 min.
6. Save Supernatant (this SN is the unbound protein) at 4°C
   1. Spin Supernatant once more to remove any excess substrate
7. Add 500 µl Buffer to pellet and mix gently with pipette tip.
8. Centrifuge at max rpm for 8 min.
9. Aspirate wash buffer from pellet.
10. Repeat Steps 7-9 twice more (3x washing step)
11. Add 200 µl of 1% SDS. Boil for 5-10 min.
12. Centrifuge for 8 min at max rpm.
13. Save pellet supernatant (this is the bound protein) andpellet.
    1. Spin pellet supernatant for 1 min @ 4°C to remove excess substrate 
       Note: Keep everything cold from this point on:
14. To the SN (step 6) and pellet SN (step13), add 100% TCA (4°C) to a final concentration of 20% TCA. Vortex.
15. Incubate overnight at 4°C.
16. Pellet at max speed for 30 min at 4° C. Aspirate SN.
17. Add 500 µl -20°C Acetone. Vortex quickly. Spin at max rpm for 20 min.
18. Aspirate Acetone. Allow to sit at RT for 10 min minimum for Acetone evaporation.
19. Resuspend pellet in 15 µl of H₂O.
20. Add 6.7 µl 6x SDS Buffer and boil for 10 min. If solution is not blue, add 2 µl Tris pH 8.8
21. Run SDS/PAGE gel.

References:

van den Burg HA, Harrison SJ, Joosten MH, Vervoort J, de Wit PJ. Cladosporium fulvum Avr4 protects fungal cell walls against hydrolysis by plant chitinases accumulating during infection. Molecular Plant-Microbe Interactions. 2006 Dec;19(12):1420-30

Tjoelker LW, Gosting L, Frey S, Hunter CL, Le TrongH, Steiner B, Brammer H, Gray PW. Structural and functional definition of the human chitinase chitin-binding domain. Journal of Biological Chemistry. 2000 Jan 7;275(1):514-20.

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