Figure 1. Sandwich ELISA analysis of complex glycans

Materials and Reagents

1. Cell wall extracts [50 mM cyclohexanediamine tetraacetic acid (CDTA) (Sigma-Aldrich, catalog number: D1383 ) or 4 M KOH/1% (w/v) NaBH₄ (Sigma-Aldrich, catalog number: 452882 ) or purified cell wall polymers]
   Notes:
   1. Reagents for cell wall extracts are 50 mM cyclohexanediamine tetraacetic acid (CDTA) (Sigma-Aldrich, catalog number: D1383) or 4 M KOH/1% (w/v) NaBH4 (Sigma-Aldrich, catalog number: 452882)
      
   2. Cell wall extraction protocol using 4 M KOH/1% (w/v) NaBH4 is described in Cid et al. (2010). This extraction will disrupt and release most cell wall glycans.
      
   3. CDTA is a chelating agent which is used to specifically extract the pectin fraction from cell walls. The same protocol can be followed using 50 mM CDTA instead of 4 M KOH (no neutralizing step necessary).
      
2. Skimmed milk powder (Marvel Original) [used at 5% (w/v) in 1x PBS]
   
3. Purified recombinant carbohydrate-binding module (CBM)
   Note: A protocol for CBM expression and purification can be found in Lee et al. (2013).
   
4. Primary monoclonal antibody (PlantProbes, www.plantprobes.net)
   
5. Secondary antibody (depending on the origin of the primary antibody used)
   1. Anti-rat IgG-horseradish peroxidase (HRP)-conjugated (Sigma-Aldrich, catalog number: A9542 )
      
   2. Anti-mouse IgG-horseradish peroxidase (HRP)-conjugated (Sigma-Aldrich, catalog number: A6782 )
6. 10 mg/ml tetramethyl benzidine (Sigma-Aldrich, catalog number: T2885 )
   
7. 1 M sodium acetate buffer (pH 6)
   
8. 6% H₂O₂ (VWR International, catalog number: 2858175C )
   
9. 2.5 M H₂SO₄ (VWR International, catalog number: 191675A )
   
10. Tween 20 (Sigma-Aldrich, catalog number: P2287 ) (optional)
    
11. 1x phosphate-buffered saline (PBS) (pH 7) (see Recipes)
    
12. HRP developing solution (see Recipes)
    

Equipment

1. 96-well surface treated ELISA microtitre plates (Thermo Fisher Scientific, Nunc-Immnuno Maxisorp, catalog number: 442404 )
   
2. Microtitre plate reader (450 nm absorbance) (Thermo Fisher Scientific, Multiskan^TM FC Microplate Photometer)
   
3. Multi-channel pipette (if available, not critical)
   
4. pH meter
   

Procedure