To calculate total cost per peptide add cost per residue plus modification plus purification. Standard immunological grade (60-70% purity) is supplied.
Standard Conjugation Protocols:For coupling through amino groups: A single or two step reaction using glutaraldehyde as a crosslinking agent is performed to link peptide to carrier (KLH in this case) via amino groups in both molecules. The most important factor is to control the crosslinker concentration, as excess reagent may cause overcoupling. The two step reaction involves activation of the peptide with glutaraldehyde followed by purification on Sephadex G-10 columns to remove excess crosslinker. The activated peptide is then reacted with carrier protein and purified on G-25 columns. For single step reaction, the peptide, crosslinker, and carrier protein are incubated together and then purified by dialysis. (Protein-protein and peptide-peptide crosslinking is possible by this method which makes it less desirable than cysteine mediated coupling). Coupling efficiencies, followed by amino froup monitorin, are in excess of 80%.
For coupling through cysteines: We use maleimidobenzoic acid-N-hydroxysuccinimide ester (MBS) as a crosslinker. The carrier protein (KLH in this case) is activated first with MBS and then free crosslinker is removed by Sephadex G-25 size exclusion columns. The activated KLH is then reacted with the cysteine containing peptide. The reaction is extremely efficient and coupling efficiencies in general are in the 80-95% range.
