Using the facility
When you wish to have peptide(s) synthesized, please contact Jill Meisenhelder for a short chat about their sequence and any modifications that are desired, and about the deprotection/cleavage step wherein the peptide is cleaved from the resin and the amino acid side chain-protecting groups are removed. You will need to provide a Salk account number for Jill to use to order the cartridges for the synthesizer and to use for the mass spec analysis of the lyophilized peptide. Depending on her schedule and yours, a time will be arranged for one of you to do the deprotection/cleavage step; in any case this can be done in the Hunter lab hood and all necessary reagents are provided.
Turn-around time will include several days after ordering the cartridges for their receipt, and another week or so for the synthesis plus the subsequent deprotection/cleavage reactions and mass spec analysis. If necessary, analytical HPLC can be arranged to further gauge the purity and composition of the synthetic peptide. For some peptides, depending on their length and application of use, HPLC purification may be necessary; this too can be arranged.
If you are going to use the peptide(s) in question as antigens you should consider the following:
- Run the sequence of your protein through a protein analysis program that will predict its secondary structure and plot predicted regions based on their hydrophobicity/hydrophilicity, surface probability, and antigenicity. One such program available on a key server at Salk is the Protean program in Lasergene Navigator. Simply create a new protein file in the Edit Seq portion of Lasergene and then open it in Protean to get an automatic full analysis! Once you’ve picked regions of high surface probability and antigenicity, enter those regions as a new sequence file to get a more detailed analysis on which to base your peptide selection.
- Check the sequences of your selected peptides against the relevant databases using the NCBI Blast program. This will reassure you that the antigen you have chosen will not produce antibodies recognizing (m)any other proteins!
- How are you going to couple the peptide to a carrier protein such as KLH or BSA? Adding a Cys residue for coupling via the SH group is commonly done; coupling can also be done through either Lys or Tyr residues.
- Where in the protein sequence does this peptide lie? If it’s at the very N-terminus you will need to couple the peptide via its C-terminal residue; if it’s at the very C-terminus of the protein you will need to couple via the N-terminal peptide amino acid. If the peptide sequence is situated in the middle of the protein you might consider a) coupling via the N-terminal residue but having the synthesis done on an amide resin so that the C terminus of the peptide will not have a non-indigenous, potentially antigenic COOH group; or b) coupling via the peptide’s C-terminal residue.