FAQ – Technical
Does the ResPep CF have to be operated in a fume hood?
No, the instrument comes with a self-contained cabinet. An exhaust tube leading to a ventilation system must be connected to an outlet provided.
How easy is a change of synthesis scale on the ResPep CF?
To select a different scale you have to load the correct synthesis plate and select a protocol corresponding to this scale. We supply optimized routines for several synthesis scales.
Is there any synthesis monitoring in the ResPep CF?
No, the MultiPep CF follows an optimized coupling protocol. Sequence-related problems must be deducted from the mass spectrum of the final peptide product.
Can I use bulk reagents?
Yes, the ResPep CF was designed for economical operation. Solvents and activator are bought from standard, recommended suppliers. Only amounts needed for a particular synthesis are weighed, dissolved and used.
FAQ – Application
What length of peptides can be made with the ResPep CF?
There is no software limitation of the peptide length. The practical possible peptide length is a function of the individual sequence and the quality desired.
How much peptide will I obtain?
The milligram yield of a synthesis on the ResPep CF is the result of the quotation: synthesis scale x molecular weight x yield. The theoretical mg amount for a typical peptide of 14 residues would be: 50 µmol x 1500 µg/µmol = 75 mg. The practical yield is usually around 50 to 80 % of this.
What is the purity of peptides made on the ResPep CF?
The purity of the final product can not be predicted as it is strongly sequence-related. Some amino acids are sensitive to the conditions of work up and some sequences form structures which interfere with the following coupling reactions. Most 12-15mer peptides are made with a purity of better than 70 %.
Do the peptides made on the ResPep CF require purification?
This depends on the application. Synthetic peptides must always be characterized by HPLC and mass spectrometry. Based on these data the purity requirements must be checked against the application. Typical immunological applications can usually work with crude material of 70-80% purity.
How long does a synthesis take with the ResPep CF?
Our pre-defined protocols have a cycle time of about 2 hours per coupling, depending on the protocol and the number of peptides actually synthesized in parallel.
Which chemistry can be used in the ResPep CF?
Our protocols were optimized with the mild Fmoc-chemistry which can be applied with less stringent safety precautions than the old Boc-chemistry.
Can the ResPep CF introduce fluorophores or unnatural amino acids?
Yes, as long as a standard coupling chemistry is being applied, any type of building block compatible with Fmoc-chemistry can be introduced. The ResPep CF employs in situ activation of carboxylic acid compounds. Up to 48 / 30 building block stock solutions can be stored on the work area for unattended coupling. Out of three different activation procedures can be assigned to individual derivatives.
What is the coupling efficiency?
Using our optimized protocols and efficient activating reagents coupling yields above 99 % can be achieved. The yields can drop significantly due to sequence-related difficulties for individual peptides.
Can a failed synthesis be optimized?
The ResPep CF manual lists several possibilities to optimize a synthesis, such as longer reaction times, double coupling protocols, or use of different derivatives. Most sequences can then be made in sufficient quality.
How do I cleave the peptides from the synthesis support?
We supply an easy instruction on peptide cleavage and workup. In short, the resin is treated with acid, peptide solution filtered off, precipitated with ether, filtered, dissolved and lyophilized.
Is there any mixing of the resin during the synthesis?
There is no mixing of the resin during coupling. The activated amino acid solution will be introduced at a high flow rate to agitate the resin bed initially and then just fills the interstitial volume between the resin beads. We use a high concentration of reagents to drive the reaction and there is enough reagent excess within diffusion range. Agitation would require a larger volume and therefore lower reagent concentration, which is less effective.
Are there any problems due to resin swelling during synthesis?
We recommend resins of the POE-PS type (e. g. TentaGel) which exhibit favourable physical properties and do not change much in volume during the synthesis. Swelling is less of a problem than shrinking, which often leads to lumpy resin beds. This can be improved by a solvent change during the cycle, which also helps to break up peptide chain aggregations causing the shrinking of the resin.
FAQ – Software
Does the ResPep CF require a computer?
The ResPep CF is operated from a standard PC. The graphical software takes care of derivative and sequence definition, generation of peptide sequences from parent protein sequences, calculation of reagent consumption, operation of the instrument and generation of a log file of operation.
How much programming is required to operate the ResPep CF?
During everyday use you just define the peptide sequences, select a protocol, load the work area and start the synthesis. More experienced users can modify the pre-defined protocols and adapt them to their specific needs. The relevant parameters of the protocol are accessible in the software.
Do I have access to all synthesis parameters?
The synthesis cycle is composed of individual tasks of the robot. The software lets you have full access to all relevant parameters and you can modify existing methods or create new procedures.
Is there a record of what the instrument does?
All instrument operations are automatically recorded to the computer hard disc. The files can be opened by standard text editors.