Overlapping peptide mixes are commercially available from vendors like Jerini Ag in Germany (http://www.jpt.com/index.htm) or BD Pharmingen (see Download peptidemix_hotlines.pdf). These are commonly designed as 15 aa in length, with 11 aa overlaps, a paradigm pioneered by Florian Kern and colleagues (see for example Kern et al., 1999). The utility of this approach, and comparison with mixes of 9 and 20 aa peptides, can be found in our JIM paper from 2001 (Link to PubMed abstract). Here are some frequently asked questions:
How many peptides can be mixed together in a single stimulation? The answer seems to be "many", maybe up to 300 or so, without significant competition for MHC sites. However, as a practical matter, peptides are usually solubilized in DMSO, and there is a limit to their solubility (determined largely by sequence). More than 0.5% final concentration of DMSO can cause toxicity to lymphocytes in a 6 hour assay. So you may be limited on how many peptides you can mix together based simply on DMSO concentration.
What purity of peptides is required? We use 80% purity as a standard, but this is more to ensure roughly equal representation of different peptides in a mix, rather than a requirement for performance. We have not been able to see a difference in stimulation capability between purified and crude versions of a single epitope peptide for CMV.
Do you order them pre-mixed? We have been able to get manufacturers to pre-mix and aliquot lyophilized mixes, and to QC these mixes for us. This is much more convenient than solubilizing individual peptides and then mixing them, and allows for higher per-peptide concentrations when dissolving the final mix. Vendors that offer pre-mixing, QC, and aliquoting include CPC Scientific (http://www.cpcscientific.com/) and American Peptide Company (http://www.americanpeptide.com/).
How do you stimulate with peptides? Brefeldin A can be added at time 0 without detriment, because internal processing of peptides is not required. 6 hour stimulation is standard, but this can be lengthened up to 12 hours or more with increasing intensity of cytokine staining, before toxicity of the brefeldin A begins to interfere.
How do 15-mers stimulate CD8 responses? We don't know for sure, but it seems likely that cell-surface and/or serum-derived proteases play a role in clipping longer peptides to generate species that can bind efficiently to class I MHC molecules. Internalization does not seem to be required, as stimulation is not inhibited by chloroquine or inhibitors of TAP processing.
What concentration of peptide is required? We typically use 1-2 ug/mL of each peptide in the mix. The optimal concentration will actually be donor-dependent, since some donors will respond to high-affinity epitopes and/or epitopes that are easily presented, while other donors will not...so this is just a compromise recommendation.