Many protein-protein interactions can be studied using a synthetic peptide as one of the partners. We make peptide arrays on cellulose membranes, which can be probed by an interacting protein to allow the study of molecular recognition events.
Typical experiments are antibody epitope mapping, protein-protein interaction studies and kinase assays.
Peptides are synthesised on a cellulose sheet derivatised to have 8-10 ethylene glycol units between the sheet and an amino group for synthesis. Loading of the amino group is about 300-400 nmol/cm2. A spot of 3 mm diameter with contain about 20 nmol of peptide.
Up to 777 spots can be synthesised on an array and peptides up to 20 amino acids long are commonly made.
Peptides have their C-Terminal anchored to the cellulose. The acetylated N-Terminal projects out from the sheet.
A possible experimental set-up is to scan a protein across its length by making 16-25 mers shifted by one amino acid, then looking for the site of interaction with its protein partner.
Once a binding site has been found using a peptide array, it is often validated by synthesising the interacting peptide(s) to perform pulldown or inhibition of binding assays.
Once binding sites are validated, mutation arrays may be synthesised in which every amino acid in a sequence is substituted by every other amino acid. This allows detection of the amino acids that are most important for a particular protein-protein interaction.
Dhira Joshi leads on synthesis of arrays.