MultiPep 1 and MultiPep 2 Frequently Asked Questions

What length of peptides can be made with the MultiPep?

There is no physical limitation of the peptide length. The practical possible peptide length is a function of the individual sequence and the quality desired. The MultiPep as a multiple peptide synthesizer was optimized for synthesis of peptides with 10 to 30 amino acids which is the typical size in biological research applications.

How much peptide will I obtain?

The milligram yield of a synthesis on the MultiPep 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: 5 µmole x 1500 µg/µmole = 7.5 mg. The practical yield is usually around 50 to 80 % of this.

What is the purity of peptides made on the MultiPep?

The purity of the final product cannot be predicted as it is strongly sequence related. Some amino acids are sensitive to the conditions of synthesis and work up. Some sequences form structures which interfere with the following coupling reactions. In cases, the protocols with TentaGel S-RAM resin gives 70% for 15-20mer peptides with natural amino acids. For peptide sequences which are parts of proteins existing in nature, 80% purity can be expected for most of the cases.

Do the peptides made on the MultiPep 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 MultiPep?

Our pre-defined protocols have a cycle time of about 3 hours per double coupling, depending on the protocol and the number of peptides synthesized in parallel.

Does the MultiPep 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.

Does the MultiPep require a computer?

The MultiPep is operated from a standard PC, WIN10. 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 MultiPep?

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.

Are ready-to-use protocols included?

All the units/options you get come with most recent and tested protocols. Automatic calculation function for reagent preparation is included in the software.

How easy is a change of synthesis scale on the MultiPep?

To select a different scale, you must load the synthesis plate or column and select a protocol corresponding to this scale. We supply optimized routines for several synthesis scales.

Can I edit synthesis method?

From the minute change of the parameters to the big change of the synthesis scheme, any editing is possible. Reagent re-calculation is done automatically in response to any change of the synthesis method.

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.

Should I buy dedicated reagent kits for MultiPep?

No dedicated kit is needed. You can use the reagents you are using in your lab. CEM offers a complete suite of peptide synthesis reagents for optimized SPPS, whether using conventional synthesis or microwave irradiation. This includes a complete library of standard and unique, high-quality Fmoc amino acids, PEG and polystyrene resins, and the powerful Oxyma Pure activator. Using CEM’s unique high-quality reagents provides the highest purity peptides, with CEM’s innovative methodology and instrumentation

Should I buy dedicated resins for MultiPep?

CEM’s SPPS resins are of the highest quality and optimized for the synthesis of standard and difficult peptides, with a variety of linkers. We developed protocols based on TentaGel S-RAM but all know SPPS resins can be used in most cases.

Can I use bulk reagents?

Yes, the MultiPep 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.

Which chemistry can be used in the MultiPep?

Our protocols were optimized with the mild Fmoc-chemistry which can be applied with less stringent safety precautions than the old Boc-chemistry. The instrument is designed to perform automated synthesis of polyamides such as peptides or PNA at a relatively small scale of a few micromoles using Fmoc-chemistry in 96-well plates or micro columns. The chemistry is optimised for Fmoc-based peptide synthesis with activation by Uronium activation HBTU/NMM or a similar protocol. The 48/72 column module performs the synthesis in individual columns at a scale of 10-300 micromoles. Reagent and solvent volumes have been well adapted to the different scales. A single probe picks up reagents and distributes them to the wells of the reaction module as defined by sequence and chemistry parameters.

Can I use Boc Chemistry on MultiPep?

No! Use of TFA significantly reduce lifespan of the unit by corrosion.

Can I apply mixing/agitation in the synthesis?

Eight column model of MultiPep1 has an orbital shaker as a standard option. Forty-eight column model of MultiPep2 also has an orbital shaker as a standard option. Seventy-two column model of MultiPep2 has an optional shaker. Other small-scale synthesis units does not have shaker options as diffusion plays major role during synthesis.

Can I perform synthesis under the nitrogen environment?

Since peptides are rarely oxidized during the synthesis, MultiPep does not have gas purge as a standard option. However, we can add a gas port, if necessary.

Can I perform cleavage on the MultiPep?

Cleavage should be done manually outside of the unit. (Use of TFA significantly reduce lifespan of the unit.) For 48 column synthesis model, dedicated cleavage unit, Cleavage Pro performs cleavage from the resin automatically.

Can the MultiPep introduce fluorophores or unnatural amino acids?

Yes, if a standard coupling chemistry is being applied, any type of building block compatible with Fmoc-chemistry can be introduced. The MultiPep employs in situ activation of carboxylic acid compounds. Up to 48 building block stock solutions can be stored on the work area for unattended coupling. Different activation procedures (Coupling type) can be assigned in the derivative table.

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.

Is there any synthesis monitoring in the MultiPep?

No, the MultiPep follows an optimized coupling protocol. Sequence-related problems must be deduced from the mass spectrum of the final peptide product.

Can a failed synthesis be optimized?

The MultiPep 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 (TFA), peptide solution filtered off, precipitated with ether, filtered, dissolved and lyophilized.

Which reaction plates are delivered with the MultiPep?

Our default scale is 5 µmol and we will deliver a basis set of 96 well reaction plates and amino acid vials for this scale. The plates are also usable at a scale below 5 µmole. The TentaGel S-RAM resin with a loading below 0.25 mmol/g was successfully used for the 5 µmol synthesis scale. A higher loaded resin can be used for a synthesis scale from 5-10 µmol.

How do we fill empty 96-well synthesis plates?

To fill the individual cavities of the reaction plate you make a slurry of resin and then pipet it into the wells.

What is the maximum practical length of a peptide that can be synthesized?

The MultiPep software supports unlimited couplings per column. Peptides of 10-20 residues can frequently be used without purification for a screening project. Longer peptides can be made but may require purification. We supply synthesis protocols with optimized double couplings for extra quality of long peptides.

Is there any mixing of the resin during the synthesis in 96-well plates?

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.

What is the recommended method for the storage of synthesized peptides

Lyophilized peptide can be stored at -80 °C for years. For use, equilibrate the peptide to room temperature in a desiccator before opening and weighing. Centrifuge the vial of the lyophilized peptides at 12,000 x g for 20 seconds before reconstitution with water. Sequences that contain cysteine, methionine, tryptophan, asparagine, glutamine and N-terminal glutamic acid will have a shorter shelf life.

How can I get the sidechain protected peptide cleaved from the resin?

Alternative to the commonly used Rink-resin use Fmoc-Ramage-Amide-Resin (Nova, Iris) from which the peptide can be cleaved with 3% TFA in DCM. Note that this resin gives C-terminal amide. For C-terminal acids use TCP-resin, cleavage with HFIP (Hexafluoro isopropanol, 25% (v/v) in DCM) results in fully side chain protected peptides.

How long can synthesized peptide be left in the room temperature (in the synthesizer) after the completion of the synthesis?

In NMP or DMF, overnight is fine. For longer time, taking the vessel out of the synthesizer and wash the resin with DCM followed by storing it dray.

How can I get side-chain unprotected peptide still bound to synthesis resin?

Use Wang resin and 25% TFA solution for sidechain deprotection. Alternatively, use Shepard resin (HMBA-AM resin) and 25% TFA for side chain deprotection.

How can I improve the yield in the small-scale filter plate synthesis?

Use Ramage resin (Nova, Iris) from which peptides are cleaved instantly with the small volume cleavage cocktail.

How can I synthesize PNA using MultiPep 1&2?

Use the following settings:
PNA monomers: 0.3M in NMP use a new coupling type
Activator: HATU or PyAOP (0.6M solution);
HATU is more stable, PyAOP should be renewed every day.
Base mix: DIPEA/2,6-lutidine (1.2 / 1.8M in DMF), mix both bases as 20.09% in DMF (v/v/v).

How can I reduce the racemization?

Around 1% racemization exists in all the synthesized peptides. Racemization makes peptides more stable which leads to the misfolding. Phenylalanine and Histidine are subjected to the racemization. Histidine can be easily racemized when it is put at 50C or higher. The combination of DIC and Oxyma Pure reduces the racemization. The combination of PyOxim and NMM is more susceptible but not as the combination of HBTU and NMM.

How can I reduce the aspartimide formation in the deprotection?

Deprotection of the Fmoc group is performed during the complete synthesis with 20% piperidine in DMF, containing 0.1 M HCOOH to avoid aspartimide side reactions.

Can I use DCM as a general solvent for peptide synthesis?

Not recommended because of the following reasons: Solubility of amino acids in DCM is not as good as in DMF or NMP Stability of an activated amino acid with “PyOxim” activator in pure DCM is less than in DMF or NMP

How can I add FITC to the synthesized peptide?

Dissolve 3 eq. FITC (Fluorescein-5-isothiocyanat) in NMP
Add 6 eq. DIPEA to the dissolved FITC. Vortex.
Add the solution to the resin-bound peptide.
Incubate overnight.
Wash with NMP and DCM
Please note: Use NOT FITC for coupling on alpha amino group, because when TFA cleavage follow, partial EDMAN degradation will be initiated. When FITC should be coupled to e.g. N-terminus of a peptide, use a linker like Ahx, etc…

How often should I change the activator solution stored in the open vial?

COMU, PyBOP, PyOxim should be renewed every day. HATU, HBTU, HCTU can stand 3 days.

My peptide has +98 MW. What does this mean and how can I avoid this?

Activator equivalent in excess compared to amino acid derivatives. A Tetera-methylguanidium termination by-product on amine was formed. Make activator equivalent or slightly less – amino acid building block always in excess.

My peptide has + 96 MW. What does this mean and how can I avoid this?

TFA was bound. Dissolving the peptide into 0.1M ammonium bicarbonate followed by lyophilisation eliminate the TFA.

How acetylated lysine can be used for the synthesis?

Get Fmoc-Lys-(Ac)-OH CAS#159766-56-0 and couple it under the same standard coupling method provided with MultiPep 1&2.

What is the hydrophilic counterpart of the Ahx linker?

Fmoc-8-amino-3,6-dioxaoctanoic acid can be used to replace Ahx linker and is recommended.

Does MultiPep requires a dedicated fume-hood?

Only a 10cm diameter duct and a 10mm PP tubing should be introduced into the fume-hood as exhaust outlets. We recommend placing the unit within 2m distance from the fume-hood, but the unit will be placed outside, and no dedicated fume-hood is needed.

Key features of the MultiPep:

• predefined and optimized chemistry protocols are coming with the machine
• large work area for more reagents
• use of amino acid stock solutions
• optimized protocols for long and difficult peptides
• parallel synthesis in plates or columns
• variable synthesis scale from 1 - 150 mg crude peptide, (1, 5, 10, 25, 50, 100 – 300 micromole synthesis scale)
• column module with no moving parts and small dead volumes
• disposable plates or columns avoid cleaning of the reaction module
• full visual control during synthesis
• easy work up in plates or columns after the synthesis
• low reagent and solvent consumption
• PC control with new optimized synthesis protocols, WIN10
• intuitive graphical software
• powerful peptide synthesis software with access to all parameters
• sequence, method and tray editor under one graphical interface
• full documentation of the synthesis parameters

How big is the MultiPep?

MultiPep 1： 580（W） x 530（D） x 720（H） mm
MultiPep 2： 890（W） x 650（D） x 790（H） mm

Does the MultiPep require additional space for the peripherals?

Addition space needed are as follows:
Space for: Solvent bottle (2L x 2 or more) beside the unit
Laptop/PC space (600 mm) beside the unit
Lifted front door (600 mm) above the unit
Waste container (10L or 20L), a condensation bottle (500mL) and a vacuum pump (300 x 500mm) under the bench.

What is SPOT synthesis?

Synthesizing a peptide array directly on a cellulose membrane (derivatized filter paper). Six hundred of peptides can be synthesized on the membrane and can be assayed simultaneously. The same assay can be done with CelluSpot which can be downsizing the assay system and can have up to 500 identical copies. CelluSpot can be recommended for mass screening of serum stocks. The SPOT method was developed by Ronald Frank for simultaneous multiple peptide synthesis on separate sites on a homogeneous membrane carrier. The principle of the t2.0echnique is to dispense small droplets of pre-activated amino acid derivatives onto a predefined array of positions on a porous membrane. The droplets get absorbed and form individual reaction compartments for chemical reaction in solid phase synthesis. A great number of distinct spots can be arranged on a sheet of membrane and each of these is individually addressable by manual or automated delivery of the respective reagent solutions. The number of spots per area and the spot size are determined by the absorptive capacity of the membrane and the volume of the drops. According to the specific functionality of the matrix, the spot size correlates with the particular scale of the synthesis. The membrane originally used by Ronald Frank is still the most popular support, made from derivatized cellulose filter paper.

What is the basic procedure?

Peptide synthesis is a cyclic procedure with several steps carried out for each amino acid addition.
Step 1: DMF wash
Step 2: Ethanol wash
Step 3: Vacuum drying of the membrane and preactivation of the amino acids
Step 4: Spotting of activated amino acids and waiting for reaction time
Step 5: Capping, optional
Step 6: DMF wash
Step 7: Fmoc deprotection
Repeat synthesis steps 1-7 until the desired peptides have been assembled.

After peptide assembly the side chain protection groups must be removed in a different protocol using a mixture of trifluoroacetic acid (TFA) with appropriate scavengers.

Do the amino acids have to be pre-activated for spotting?

No, the MultiPep 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.

Which chemicals are provided by CEM?

We offer bulk Fmoc amino acids in vials. They are activated by the DIC/Oxyma Pure method. Other chemicals, such as, solvents and reagents must be acquired from a local source. We provide a list of chemicals needed and sources for them. The chemicals should be bought well before installation of the MultiPep to allow starting a real synthesis run during the training session.

How are the data from SPOT synthesis evaluated?

Detection is performed mostly by labelled probe methods, such as fluorescence, chemiluminescence, electrochemiluminescence and radioactivity detection. Deposition and simple densitometry using a flatbed scanner, or by exposure of a film if e.g. radioactive phosphorus has been incorporated. Chemiluminescence works well for most cases, but fluorescence can be used for very low affinity interactions since it is a more sensitive method. 　Since cellulose has the autofluorescence of visible light range, we recommend infrared labelling. Detection and data handling are standard techniques which we do not support.

What can be done with the MultiPep spot robot?

The MultiPep Robot has been developed to reliably deliver amino acid derivatives to synthesis arrays on membranes. A pipetting robot under the control of a WIN10 PC based software now delivers volumes down to 100 nL to up to a few thousand individual spots. Four membranes of microtiter plate format can be mounted on the work area. Standard grids are 384 or 600 positions, but densities of up to about 1,000 spots per membrane have been achieved. The regular grid can be freely defined. Up to 48 or more amino acid derivatives can be used which allows to use natural and non-natural amino acids in one run.

Are the membranes deprotected in the instrument?

Yes, the MultiPep 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 since it is a fully automated peptide synthesis system.

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 during the preactivation of the next building block is being applied to remove ethanol 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. It is even more effective than washing manually in plastic trays.

Can we still use Bromophenol blue (BPB) to monitor the reactions?

The MultiPep 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 more than 600 spots on each membrane?

So far, we have worked only with 384 and 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.

What is the synthesis time for two sets of 600 each 10-mer peptides?

With a recommended double coupling of 2x30 min, 2x5 min deprotection and all washing and drying steps, the total cycle time is about 3 hours and the two sets of 10mers would be assembled in a little more than a day.

Can the synthesis be interrupted and started again?

The MultiPep 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.

What is the density of peptides on the membrane?

The standard density is a grid of 600 peptides spots with 4.2 mm centre-to-centre distances. Other grids are possible and can be freely defined. Densities of up to 25x40 spots on a cellulose membrane have been achieved under very controlled conditions.

How much peptide is in a spot?

The membranes we supply are derivatized to a loading of about 300-400 nmol/cm² free amino groups. The loading per spot can be calculated from the area using this value. For example: A spot of 6 mm diameter would have about 80 nmoles, a spot of 3 mm diameter about 20 nmoles of peptide.

What type of computer is required?

The SPOT synthesis software will run on almost any PC under Windows 10. The PC used must not be networked or used for other applications at the same time as this would slow down the operation. A standard laptop is part of the shipment.

What are the features of the software?

As automation of the SPOT synthesis procedure with the MultiPep allows to handle large arrays, a powerful software is necessary to generate the sequences. Sequences can be entered in the software itself or imported from text files in one letter code from other sources. There are several modes to generate peptides from parent protein or peptide sequences:

• individual peptides (entered one by one)
• analogue sequences (by defined amino acid replacement)
• overlapping peptides (generated from a protein sequence)
• overlapping peptides of different lengths (from a protein sequence)
• peptide libraries with a maximum of two mixed positions (libraries)
• Alanine walks, 2 position libraries
• N- and C-terminal truncation
  Sequences are entered as one letter code (OLC) or multiple letter code (MLC). The instrument can handle as many amino acid derivatives as you like, provided derivative positions are available. This allows use of natural and unnatural amino acids in one synthesis run. The maximum length is not limited by the software, but practically the sequences should not be longer than 15-30 residues for Spot synthesis project.

Which chemistry is used?

The synthesis is carried out with Fmoc-protection chemistry on membranes made of pure, derivatized cellulose. The amino acid building blocks are derivatives which are protected at their amino terminus by 9-fluorenyl-methoxycarbonyl (Fmoc). The protection group ensures that in each step only a single building block is coupled to each growing peptide chain. Trifunctional amino acids also carry a side chain protection group. The Fmoc group must be removed in each synthesis cycle whereas the side chain protection groups are taken off at the end of the synthesis. In situ activation of the amino acid derivatives is performed by DIC / Oxyma Pure, which leads to rapid activation and coupling. A membrane can be used to synthesize many individual peptides which can be screened for biological activity by a Western-blot-like assay. Alternatively, the spots are cut out and cleaved separately from the support if a suitable linker had been introduced prior to the synthesis.

Is it possible to make soluble peptides with the MultiPep?

Several people have worked on cleaving peptides from the SPOT membrane with different degrees of success. The major problem was always to get rid of the side chain protection groups while having the peptides still attached to the cellulose. Yes, but you would need to add a cleavable linker on the paper. Or using the beta-Ala membrane you can cleave as well but the C-terminus is modified. Cleavable peptides are better if they come from a resin synthesis – 96 well filter plate. Suitable resins are available.

Is there a specific protocol to be used in the synthesis of peptide spots?

There is a detailed protocol in the manual describing the chemistry and the derivatives to be used. There are also other protocols in the scientific literature on various applications of the SPOT technique. We provide a literature list on request.

Do you have specific protocols used for the membrane detection assays?

We do not provide information on these aspects as there are too many different assays and very specific know-how on how to use them. A lot of this is met by the literature and the literature list exceeds 600 papers now. Our manual concentrates on how to set up the instrument, define peptide sequences to be made, prepare the chemicals, run the synthesis and cleave offside chain protection groups. It ends with the peptide arrays on the membranes ready for your assay.

Do you provide protocols for membrane regeneration?

To reuse the membranes, they must be stripped from the protein already bound to them in the first experiment. Little is known about the effect of the stripping, regeneration and storage conditions on the array performance. Generally, the membranes should be dried and regenerated for storage, In cases where the stripping is not complete it would not be possible to reuse the membrane. We do not provide specific protocols for assays and membrane regeneration. Please refer to the literature on specific assays.

How is the peptide synthesis reliability (purity, amount) validated?

There is no validation except for comparison with reference sequences to be defined by the user for his respective assay. It is very difficult to check the purity and quantity. There are a few literature references to procedures used. In general, you should work out internal references and standards for your assay.

What are the chemistry limitations regarding the peptide length?

We recommend peptide lengths of no more than 10-25 residues. It has been shown that the synthesis quality drops sharply after a chain length of 25 residues, probably due to sterically reasons. Due to the open synthesis conditions severe oxidation of cysteine and methionine must be expected.

What is the chemical stability of the membranes?

The membranes used are based on acid hardened cellulose and derivatized with a polyethylene glycol spacer. This spacer is stable in a pH range of 1-14. The paper itself is stable to the synthesis conditions but should not be exposed to strong acid for prolonged periods. The cleavage by dilute trifluoroacetic acid for 1-2 hours is the limit of chemical stability.

Are peptides lost by repeated stripping?

The original protocol published is based on derivatization of the paper with beta-alanine by an ester bond. This bond was cleaved under slightly basic conditions, e. g. most of the peptides would be lost over the course of some hours at pH 8. The new PEG spacer introduced in 1998 eliminates this problem. There may still be users who make the derivatized membranes themselves according to the old published protocols which will create the problems mentioned.

What is the loading of the membranes?

The membranes we supply are derivatized to a loading of about 300-400 nmol/cm2 free amino groups on PEG spacers of about 500 Da molecular weight.

How are your membranes validated?

The membranes are manufactured according to standardized protocols with some quality control steps along the way. The loading in terms of amino functions available is determined by bromophenol blue binding and photometric determination.

What kind of membranes are commercially available?

We deliver only one sort of membrane which is cellulose derivatized with amino functions via an acid and base stable PEG spacer.

Is it possible to synthesize PNA arrays with the MultiPep?

PNA is a very interesting DNA analogue based on amino acid like building blocks. PNA spot arrays can be used for DNA hybridization. For protocols, please refer to papers published by Jörg Hoheisel et al.

Can the MultiPep be used to make fluorescently labelled peptides?

Some users have made fluorescently labelled peptides and coupling of Rhodamine to some control sequences is also a nice way to monitor the synthesis. However, due to the open handling we would expect problems with photobleaching.

Can the peptide SPOT arrays be used for protease assays?

Protease assays have been very difficult and only a very small fraction of the peptide on the paper is accessible for cleavage by enzymes. If is not possible to measure disappearance of labelled material but only the cleaved products themselves. Therefore, the spots must be cut out before the assay which is quite cumbersome.

Can the peptide SPOT arrays be used for kinase assays?

Kinase assays are an ideal application for peptide SPOT arrays as the labelled paper can be exposed easily to X-ray film for evaluation. There are several papers in the literature about this kind of assay.

Key features of the MultiPep SPOT Synthesis:

• predefined and optimized chemistry protocols are coming with the machine
• large work area for more reagents
• use of amino acid stock solutions
• optimized protocols
• parallel synthesis on 2 membranes, up to 2x600 peptides, MultiPep 1
• parallel synthesis on 4 membranes, up to 4x600 peptides, MultiPep 2
• full visual control during synthesis
• easy work up after the synthesis
• low reagent and solvent consumption
• PC control with new optimized synthesis protocols
• intuitive graphical software
• powerful peptide synthesis software with access to all parameters
• sequence, method and tray editor under one graphical interface
• full documentation of the synthesis parameters

What is CelluSpot synthesis?

Array of cellulose-bound peptides on coated microscopic slides. A duplicate of 384 peptide spots is on the slide. Peptides are synthesized on cellulose disks (like the SPOT synthesis) followed by dissolving the disks and printed on the slides. Massive assay can be done as one synthesis can give up to 500 identical copies of the peptide array slides. CelluSpot can be recommended for mass screening of serum stocks.

What kind of assay can be done with SPOT synthesized array?

Major use of the SPOT array are as follows:
Peptide-antibody interaction
Peptide-receptor interaction
Peptide-enzyme interaction
Peptide-protein interaction
Peptide-microbe interaction
Peptide-metal interaction
Peptide-DNA interaction
Peptide modification (artificial peptides) assay
*SPOT reference list is available on request.

Is there any way to avoid interference between peptide molecules packed in high-density on the SPOT array?

Blocking a part of amino residues on the membrane reduces the density of the peptide molecules and solves the problem. Adding spacers also help.

What is bound to the membrane used for the SPOT synthesis?

PEG spacers are attached to the cellulose molecules followed by NH2.

Is there any way to test the purity of the SPOT synthesized peptides?

Place a TFA labile linker at C terminus of the peptide and cleave the peptide after the synthesis followed by MS analysis. Fmoc-Rink liker from Navabiochem can be recommended.

Is there any way to check the success of the amino acid coupling after/in the SPOT synthesis?

Staining N terminus amino residue using BPB can help. BPB confirms the existence of the terminal NH2. Alternatively, synthesized peptides can be visualized with 254nm UV light. Illuminating the membrane with UV reveals autofluorescence of peptides as bright spots. Please note that both intensity of the bound BPB as well as UV are strongly dependent on the sequence, hence quantitative determination cannot be done.

How can I store the synthesized SPOT membrane?

Skipping final deprotection and TFA cleavage leaves all the protecting groups on the synthesized peptides. Storing this membrane at minus 20 C in the heat-sealed bag retains the peptide intact for long period. Be sure to allow the membrane to be room temperature before breaking the seal of the bag. Fmoc-deprotection and side chain deprotection should be performed before use.

Non-specific binding of antibody is observed in the assay of Cys containing peptides on the SPOT membrane. Is there any way to eliminate this?

Cysteine is known to give unexpected results in many assays, in addition to its nature of making disulfide bond. Replacing Cys with Ser sometimes gives stable results.

Which brand of the filter paper should be chosen for putting it under the frame during CelluSpot synthesis?

Whatman 540 or 541 is recommended

What is “consensus sequence” mentioned in the CelluSpot Kinase array peptide list?

The consensus sequence is that sequence of amino acids which overall deviates the least from a given set of corresponding pattern sequences. They are common sequences uses for positive/negative control in Y and/or S/T kinase array.

How can I cleave the peptide from SPOT membrane?

Use Fmoc-Rink-Linker as the first amino acid. Cut the desired spot out and apply cleavage solution. The Rink-Linker cleaves itself and gives C-terminal amide.

A portion of the N-terminal amide in my SPOT peptide is acetylated. How can I avoid this?

Use Boc-amino acid for last amino acid. This will avoid acetylation by the trace amount of acetic anhydride in the filter paper and channel below it.

How long can I keep the NMP-dissolved amino acid?

Except for Cysteine and Methionine, amino acid solutions can be stored at 4°C for up to one month. Please note that Alanine and Glutamic Acid can make precipitation if the powder is deteriorated.

How can I synthesize branched peptides?

Use Fmoc-Lys(ivDde)-OH, CAS No.: 204777-78-6. The Fmoc group can be removed selectively with piperidine the ivDde protection group is cleaved with 2% hydrazine in DMF.

What amount of peptide can I synthesize on the MultiPep?

MultiPep1
Plates = up to 384 (4 x 96) peptides at 1-10 μmol / 2 mg – 10 mg

Columns = 48 micro (0.5 mL) or 24 mini columns (1.0 mL) ( 1-15μmol / 2 mg - 20 mg)
    8 columns (2,5,10 mL) - 10-100μmol

SPOT Synthesis = up to 2400 peptides on four cellulose membranes
CelluSpotsTM = up to 768 peptides on a dissolvable cellulose support for spotting on many identical slides

Mulitipep2
Plates = up to 384 (4 x 96) peptides at 1-10 μmol / 2 mg – 10 mg

Columns = 48 micro (0.5 mL) or 24 mini columns (1.0 mL) ( 1-15μmol / 2 mg - 20 mg)
    48 columns (2,5,10 mL or 12 columns 20 mL) - 10-500μmol
    72 columns (2,5 mL or 36 columns 10 mL) - 10-300μmol

SPOT Synthesis = up to 2400 peptides on four cellulose membranes
CelluSpotsTM = up to 768 peptides on a dissolvable cellulose support for spotting on many identical slides

What is the maximum number of peptides synthesized on the MultiPep?

MultiPep 1： 8 (100 μmol) / 24 (15 μmol) / 96 (10 μmol)
1200（SPOT) / 768 (CelluSpot)
MultiPep 2: 48 (100 μmol) / 72 (50 μmol) / 384 (10 μmol)
768 (CelluSpot) / 2400 (SPOTs)