FAQ – Technical
Does the ResPep SL 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.
Can I use bulk reagents?
Yes, the ResPep SL 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
Do the amino acids have to be pre-activated for spotting?
No, the ResPep SL can activate amino acid derivatives specifically for each cycle. There is a dedicated activation vial for each amino acid. Of course, pre-activated amino acids can also be used.
Are the membranes deprotected in the instrument?
Yes, the ResPep SL spots piperidine solution on the membrane to take the Fmoc protection group off in each cycle. The membrane can stay in the instrument for the entire synthesis.
How is the membrane washed and dried for the next coupling?
The membrane is clamped tightly in a holder which forms a shallow tray above it. The bottom has tiny holes to remove solvent by vacuum aspiration. Solvent is delivered on the upper face of the membrane and pulled through by vacuum. A longer extraction time is being applied to remove methanol from the final wash and to dry the membrane.
Is the rinsing action sufficient if a full set of peptides is built on the membrane?
Yes, the flow-through mode of washing is very effective and makes economic use of the solvents. Actually it is even more effective than washing manually in plastic trays.
Can we still use Bromophenol blue (BPB) to monitor the reactions?
The ResPep SL can be programmed to deliver BPB at the appropriate steps in the cycle to stain amino groups on the peptides assembled. The stain is removed with the next coupling reaction as it is done in manual procedure, too.
Is rinsing only applied on the spot where reagents were delivered?
No, the entire membrane must be washed, even if there is only one single spot to be coupled. In general, the peptides will be of similar length and a significant portion of the membrane is still being spotted.
Is there any possibility of piperidine carryover?
As piperidine is used in a very large excess it must be removed thoroughly. The washing procedure is designed to remove all traces of the reagent before the next coupling will be started.
Can the MultiPep be adjusted to make 1536 spots on each membrane?
So far, we have worked only with 600 spots per membrane which is relatively easy. Very dense arrays may require manual pre-activation (once a day) as the very fine needle tip is not suited for the mixing necessary in the activation procedure.
Is it possible to use a Boc-Lys(Fmoc)-Pro linker and cleave diketopiperazine peptides for QC/QA?
Yes, this can be done and we can supply the respective membrane.
What is the synthesis time for two sets of 600 each 10-mer peptides?
With a recommended double coupling of 2×30 min, 2×5 min deprotection and all washing and drying steps, the total cycle time is about 2.5-3 hours and the two sets of 10mers would be assembled in a little more than a day.
What is the cost of consumables and solvents?
Amino acid derivative sets and membranes are similar to those used in the semi-automated SPOT synthesis on our AutoSpot SL robot. The amount of piperidine used is considerably less (it accounts for most of the overall costs!) and the volume of solvents is also a bit less than in the manual procedure.
Can the synthesis be interrupted and started again?
The ResPep SL can be stopped and re-started at any time. Several convenient options allow manual procedures (e. g. Bromophenol blue staining or introduction of very unusual derivatives) and safe re-starting in any cycle.
FAQ – Software
Does the ResPep SL require a computer?
The ResPep SL 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 SL?
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.