Materials and Reagents

1. Plant material harboring the receptor of interest in an immobilized form
   Suggested plant materials could be: Intact cells from suspension cultures, ground plant tissue or isolated receptors immobilized on immunoprecipitation beads.
   Notes:
   
   1. The receptor must be immobilized (on cells, cell debris, IP-beads) in order to allow efficient washing to remove unbound ligand without washing away the receptor molecules.
      
   2. Levels of some receptors are exceedingly low. Therefore, overexpression, e.g. by transient expression in Nicotiana benthamiana (N. benthamiana) leaves (Li, 2011), may be required to obtain sufficient binding sites.
      
   3. Use a negative control, same material lacking a functional receptor, whenever possible.
2. MES (2-ethanesulfonic acid)
   
3. NaCl
   
4. DTT
   
5. 33 µl proteinase Inhibitor (Sigma-Aldrich, catalog number: P9599 )
   
6. 5 mM citric acid
   
7. 0.03 % H₂O₂ in 100 mM NaOH (prepare fresh from a 30% H₂O₂ stock)
   
8. Appropriate binding buffer (see Recipes)
   

Equipment

1. Mortar and pestle
   
2. Table top centrifuge
   
3. Luminometer (e.g. single tube machines like FB 12, Berthold technologies, that allows injection)
   
4. Acridinium-labeled peptide
   Important: The modified peptide should be tested for biological activity in your usually used bio-assay of choice and should not deviate much from the activity of the unmodified peptide.
   
5. Unlabeled peptide (synthetic unlabeled peptides can be ordered by e.g. Biomatik)
   
6. Peptides and conjugation with acridinium
   Note: Peptides can be ordered (on resin) from a company of choice or synthesized using solid-phase technology with Fmoc-protected amino acids. Acridinium esters can be conjugated to the N-terminal amino groups of the peptides on resin by coupling with N-hydroxysuccinimide activated acridinium esters (Cayman) before deprotection and purification of the peptides via HPLC.
   

Procedure