Peptide Synthesis at KI Biopolymers Lab
Welcome to Peptide Synthesis. If you have not used us before for peptide synthesis feel free to email/talk to someone at the lab because we need to know things like your desired amount, N and C terminal chemistry, if HPLC purification is needed or if you want any modifications such as dyes, biotin, lipids, etc. See below for a list of recent mods to get an idea of what you can do.
- N- & C-terminal Chemistry
- Modified Residues
- Dye Labeling
- Desalting
- HPLC Purification
- Quality Control
- Antibody Production References
N- & C-terminal Chemistry
Standard N-terminal chemistry is either free amine or acetylation, and standard C-terminal chemistry is either free acid or amidation. You may want to calculate the isoelectric point (pI) of the sequence and consider the pH of the buffer that you will be working in prior to choosing terminal chemistry. By blocking the terminals, you may end up with a peptide that is not very soluble in your working conditions. However, you may want to neutralize the terminal(s) to mimic the appearance of the peptide in vivo for better antibody recognition. See the Antibody Production Notes and References section.
Modified Residues
Modified residues, such as unusual amino acids, can be incorporated into a synthesis. If we do not have a residue in the lab, we will order it, and the user will be charged for the price of the order.
Biotin can be added to the N-terminus of a peptide or at a Lysine residue. For large scale peptides this procedure is done post-synthesis, but while the peptide is still attached to the resin. For small scale peptides, biotinylation is done on the peptdie synthesizer. Cleavage will then follow.
Common Modifications:
- N-terminal Biotin
- Fluorescein (5.6 FAM)
- TAMRA
- FMOC-Lys (Dde)
- Removal of DDE fro DDE Lys
- Phospho-tyrosine
Click here for some less common modifications.
Dye Labeling
We can attach dyes to peptides, either at the N-terminus or at a Lysine residue. The most common dyes used are fluorescein (absorbs @490nm) and TAMRA (absorbs @540nm). This procedure is done post-synthesis, but while the peptide is still attached to the resin. Cleavage will then follow.
Desalting
The peptide fraction of the crude material consists of the ideal peptide, traces of deletion peptides and modified peptides created during side chain deprotection and cleavage from the starting resin. Since purification is time consuming and not always necessary, we recommend that you experiment with the crude or desalted product before requesting HPLC purification. Most researchers use the crude peptide for coupling to carrier protein for immunization. We do not do desalting in the laboratory, but it can be done by gel filtration chromatography (e.g. G10 or G25 columns). Disposable gel columns are available from companies like BioRad and Pharmacia/Amersham. For larger scale desalting the packing material can be purchased separately and you can pour your own column. Desalting protocols are available from manufacturers catalogues and from peptide synthesis reference books such as Stewart and Young's "Solid Phase Peptide Synthesis."
HPLC Purification
If high purity peptide is required, we offer HPLC purification of peptides.
For small scale peptides, HPLC purification consists of taking 5umol of peptide and injecting that material onto a preparative scale reverse phase column. We collect all column fractions and pool those containing the ideal product. The amount of pure material recovered depends on the sequence but usually is better than 50% of that loaded.
For large scale peptides, HPLC purification consists of taking approximately 100 mg of crude peptide (the amount depends on the complexity of the analytical HPLC trace of the crude product) and injecting that material onto a preparative scale reverse phase column. We collect all column fractions and pool those containing the ideal product. The amount of pure material recovered depends on the sequence but usually is better than 50% of that loaded. The pure peptide is provided along with any crude peptide remaining from the purification process.
Quality Control
For small scale peptides, we provide you with mass spectrometry data and, upon request, HPLC data. Usually with the small scale peptides, the mass spectrometry data is a sufficient quality control. MS and HPLC are performed under high resolution/slow gradient conditions.
For large scale peptides, we provide you with an HPLC trace of the peptide to assess purity by UV along with mass spectrometer spectra. HPLC and MS are performed under slow gradient / high resolution conditions. If you have a peptide from an outside source and are suspicious about quality we will be happy to subject an aliquot to our QC procedure.
