Synthesis and Probing of Membrane-bound Peptide Arrays

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Synthesis and Probing of Membrane-bound Peptide Arrays

Ronald Frank
Department of Chemical Biology, GBF (German Research Center for Biotechnology), 38124 Braunschweig, Germany
Stefan Dubel
Institute for Biochemistry and Biotechnology, Technical University of Braunschweig, D-38106 Braunschweig, Germany
Excerpted from Protein: Protein Interactions, Second Edition
Edited by Erica A. Golemis and Peter D. Adams

ABSTRACT

The following protocol describes the synthesis of short linear peptides, or peptide pools, on modified cellulose membranes, and the detection of their protein-binding partners. Peptides are synthesized from their carboxyl termini using Fmoc-amino acid derivatives. After completion of the synthesis and cleavage of all side-chain protecting groups, the peptide array on the membrane is incubated with the potential interaction partners to identify their target sequences.

MATERIALS

Buffers, Solutions, and Reagents

Buffer salts and ingredients should be of biochemical grade.
Bromophenol blue indicator (BPB), 10 mg per ml in N,N-dimethyl formamide (DMF)
- Maintain stock solution at room temperature. This stock should have an intense orange color and should be discarded if the color has turned to green.
Acetic acid
DMF
- This should be free of contaminating amines and thus of the highest affordable purity. Purity can be checked by adding 10 µl of BPB stock to 1 ml of DMF. If the resulting color is yellow, the batch can be used without further purification. Check each new batch!
1-Methyl-2-pyrrolidinone (NMP)
- For use in the preparation of Fmoc-AA stock solutions, NMP should be of the highest purity available. Amine contamination can be checked as for DMF. If the resulting color is yellow, the NMP can be used without further purification. Most commercial products, however, are not acceptable. To purify an unsuitable batch, treat 1 liter of NMP with 100 g of acid aluminum oxide overnight under constant vigorous shaking at room temperature. The next day, retest the purity; a 1-ml aliquot should give a yellow BPB test. Filter the slurry through a bed of dry silica gel (for flash chromatography, Mallinckrodt Baker BV) in a closed glass filter funnel (slight nitrogen pressure can speed up the process, but is not necessary). Divide the clear liquid into 100-ml portions and store tightly closed at -20°C.
N-Hydroxybenzotriazole (HOBt)
- Used in the preparation of Fmoc-AA stock solutions. Anhydrous (ISOCHEM). Store tightly closed at room temperature in a dry place.
DIC (N,N´-Diisopropylcarbodiimide), =98%
Fmoc-AA stock solutions
- Fmoc-amino acid derivatives of all 20 L-amino acids as well as -alanine and other special amino acid derivatives are available from several suppliers in sufficient quality. Side-chain protecting groups should be Cys(Acm) or Cys(Trt), Asp(OtBu), Glu(OtBu), His(Trt), Lys(Boc), Asn(Trt), Gln(Trt), Arg(Pmc), Ser(tBu), Thr(tBu), Trp(Boc), and Tyr(tBu). HOBt-esters of these amino acid derivatives must be prepared in NMP for use throughout in spotting reactions. Dissolve 1 mmole of each Fmoc-AA in 5 ml of NMP containing 0.25 M HOBt to give 0.2 M Fmoc-AA stock solutions. These are kept in 10-ml plastic tubes that are closed tightly, flash-frozen in liquid nitrogen, and stored at -70°C. For use in coupling reactions with amino acid mixtures at randomized positions in the peptide sequences, combine equal aliquots of Fmoc-AA stock solutions for the respective amino acids to be incorporated, dilute with twofold volume of NMP to give 66 mM solutions, and store as described above.
Special chemical derivatives
- Free thiol functions of cysteine may be problematic because of post-synthetic uncontrolled oxidation. To avoid this, you may replace Cys by Ser, Ala, or -aminobutyric acid (Abu). Alternatively, choose the hydrophilic Cys(Acm) and leave protected. For the simultaneous preparation of peptides of different size with free amino termini, couple their terminal amino acid residues as αN-Boc derivatives so that they will not become acetylated during the normal elongation cycle. Boc is then removed during the final side-chain deprotection procedure.
Acetylation mix
- 2% solution of acetic anhydride (=99.5%) in DMF
Piperidine mix
- 20% solution of piperidine (=99%) in DMF
  
  Piperidine is highly toxic and should be handled only with gloves under a hood!
Methanol or ethanol, technical grade (95%)
Deprotection mix
- trifluoroacetic acid (TFA, synthesis grade), triisobutylsilane (TIBS), water, and dichloro methane (DCM) in a ratio of 80% TFA, 3% TIBS, 5% water, and 12%
  
  DCM
  - Trifluoroacetic acid is very harmful and volatile, and should be handled with gloves under a hood!
Tris-buffered saline (TBS)
- 8.0 g of NaCl, 0.2 g of KCl, and 6.1 g of Tris-base in 1 liter of water Adjust pH to 7.0 with HCl ; autoclave and store at 4°C.
T-TBS: TBS buffer plus 0.05% Tween-20
Phosphate-buffered saline (PBS)
- 8.0 g of NaCl
  0.2 g of KCl , 1.43 g of Na₂ HPO₄ ・2H₂ O, and 0.2 g of KH₂ PO₄ in 1 liter of water
  Adjust pH to 7.0 with HCl ; autoclave and store at 4°C.
Citrate-buffered saline (CBS)
- 8.0 g of NaCl, 0.2 g of KCl , and 10.51 g of citric acid (x1 H₂ O) in 1 liter of water
  Adjust pH to 7.0 with NaOH ; autoclave and store at 4°C.
Membrane blocking solution (MBS)
- Mix 20 ml of casein-based blocking buffer concentrate (No. SU-07-250; Sigma-Genosys), 80 ml of T-TBS (pH 8.0), and 5 g of sucrose; the resulting pH will be 7.0; store at 4°C.
Alkaline phosphatase (AP)-conjugated detection antibodies
AP-conjugated streptavidin
Color developing solution (CDS)
- Dissolve 50 mg of (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in 1 ml of 70% DMF/30% water; store at -20°C. Dissolve 60 mg of 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (BCIP) in 1 ml of DMF ; store at -20°C. Always prepare fresh CDS on the day of the experiment: To 10 ml of CBS add 50 µl of 1 M MgCl₂ , 40 µl of BCIP, and 60 µl of MTT.
  
  Never use NBT (4-nitro blue tetrazolium chloride) instead of MTT, as the developed color will not be removable from the membrane
Immun-Star Chemiluminescent Kit
Horseradish peroxidase (HRP)-conjugated detection antibodies
Chemiluminescence detection kit
Transfer buffer (for western blotting)
- 25 mM Tris-HCl (pH 7.6), 192 mM glycine , 20% methanol , 0.03% sodium dodecyl sulfate (SDS)
Stripping mix A (SM-A)
- 8 M urea , 1% SDS in PBS; store at room temperature. Add 0.5% 2-mercaptoethanol prior to use and adjust pH to 7.0 with acetic acid.
Stripping mix B (SM-B)
- 10% acetic acid, 50 % ethanol, and 40% water; store at room temperature

Special Equipment

All equipment used for membrane synthesis and regeneration should be resistant to organic solvents. Glassware or polypropyleneware should be exclusively used in all steps involving organic solvents. Standard micropipetting tips (Gilson, Eppendorf) can be employed.
3MM paper (Whatman)
Nitrocellulose membrane suitable for electro-transfer (e.g., Protan Nitrocellulose Transfer Membrane; Schleicher & Schuell)
SPOT membranes
AC-S01 type amino-PEGylated membranes (AIMS-Scientific-Products GmbH) are recommended and available from several suppliers. Ready-to-use membranes in an 8 x 12 format with 96 spots of Ala anchors are available from Sigma-Genosys.

The ASP222 instrument requires a special format of membrane with perforations for the holder pins on the robot! These are available only from Intavis AG.
SPOT synthesis kit: A kit that includes all necessary items for manual SPOT synthesis is available from Sigma-Genosys.
Software for the generation of peptide lists and pipetting protocols is included in the Sigma-Genosys synthesis kit and the operation software of the spotting robot. A freeware package is also available from the authors (frank@gbf.de).
Flat reaction/washing troughs with a tightly closing lid made of chemically inert material (glass, Teflon, polypropylene) with dimensions slightly larger than the membranes used
Spotting robot, model ASP222, or MultiPep peptide synthesizer with spotting tray (Intavis Bioanalytical Instruments AG)
Microfuge tubes, 1.5 ml (e.g., Eppendorf, safe twist) and appropriate racks as reservoirs for amino acid solutions
Rocking platform
Dispensers, adjustable from 5 ml to 50 ml for DMF and alcohol containers
Hair dryer, hand-held with cold-air function
Polystyrene plates (12 x 12 cm) with covers, as used in cell culture
Flat glass tray to hold at least one membrane
Sonication bath with temperature control
Digital recording device
Scanner or CCD camera for documentation of signal patterns on membranes plus analysis software for quantification of signals. In case of radioactive or chemiluminescence detection, autoradiography films can be used.
Plastic bags and sealing device
Blotting apparatus; e.g., Biometra-Fast-Blot
Pencil (lead type H or 2H) for marking membranes

METHOD

All volumes given below are for one standard AC-S01 paper sheet of 8 x 12 cm and must be adjusted for more sheets, or other paper qualities and sizes. Unless otherwise stated, washes and incubation steps are carried out in sealed troughs at room temperature with gentle agitation on a rocking platform. Solvents and solutions are decanted after the time indicated. During incubations and washes, the troughs are closed with a lid.

Stage 1: Preparative Work

Generate a list of peptides to be prepared. Multiple lists can be combined on a single membrane to fill up a complete array. The peptides can be separated after synthesis by simply cutting the membrane into appropriate sections.
Cutting lines between sections can be marked out on the membrane in pencil.
Select the appropriate array(s) for the chosen experiment according to number, spot size, and scale required.
For manual spotting, use an 8 x 12 format (spot distance, 9 mm; spot volume, 0.5 µl for array generation, 0.7 µl for elongation cycles). An array of 17 rows with 25 spots each (spot distance 4 mm, volume 0.1 µl for array generation and 0.2 µl for elongation cycles) is recommended for the ASP22.
Calculate the required volumes of Fmoc-AA solutions for each derivative and cycle.
Remember that a triple coupling procedure may be necessary, and that each vial should contain a minimum of 50 µl. Consider, for example, a list of peptides that requires alanine for 26 peptides at cycle 1 on a 17 x 25 array. This cycle will require 26 (peptides) x 0.2 (µl per elongation) x 3 (couplings) = 15.6 µl of Fmoc-Ala stock solution. Therefore, you will take the minimum of 70 µl of stock solution for this vial. The SPOT software can perform this calculation.
Label a set of 1.5-ml microfuge tubes with derivative and cycle code (e.g., A1), and distribute the Fmoc-amino acid stock solutions according to the calculated volumes required. Snap-freeze in liquid nitrogen and store at -70°C.

Stage 2: Generation of the SPOT Array

Mark each membrane used with a pencil label (e.g., a number or letter at the right bottom edge) for proper orientation and tracking throughout the synthesis process. For manual synthesis, mark the spot positions on the membranes with pencil dots and place the membrane in the reaction trough. For automated synthesis, fix membranes on the platform of the spot robot.
Take a 100-µl aliquot of the Fmoc-βAla stock from the freezer and bring it to room temperature. Add 1 µl of BPB stock and 4 µl of DIC. Mix, and leave for 30 min. Spot aliquots (0.5 µl for 8 x 12 array, or 0.1 µl for 17 x 25 array) of this solution on all positions of the chosen array configuration. Cover the membrane with glass plates and allow the reaction to proceed for 60 min. For peptides longer than 20mers, reduce the loading of the spots by applying a mixture of the Fmoc-βAla stock and an N-acetyl-alanine stock (1:9). This will avoid molecular crowding.
Wash each membrane twice in 20 ml of acetylation mix; once for 30 sec, and once for 2 min. Incubate the membranes overnight in acetylation mix.
Wash each membrane in 20 ml of DMF (3 times for 10 min each).
To remove Fmoc blocking groups, incubate the membrane for 5 min in 20 ml of piperidine mix.
Wash each membrane in 20 ml of DMF (4 times for 10 min each).
Visualize the spots by incubating each membrane in 20 ml of DMF containing 1% BPB stock.
Spots should be stained only light blue! If traces of remaining piperidine on the membranes turn the liquid dark blue, renew the staining solution and continue the staining.
Wash each membrane in 20 ml of methanol or ethanol (2 times for 10 min).
Transfer the membranes to 3MM paper folders and dry them using cold air from a hair dryer. Store dried membranes in a sealed plastic bag at -20°C.

Stage 3: Assembly of Peptides on SPOTs

Take the membranes from step 9 and, for manual synthesis, number the blue spot positions with a pencil (according to the peptide lists). Place the membranes in separate reaction troughs. For automated systems, fix the unmarked membranes on the platform of the synthesizer. If necessary, cutting lines should be marked in pencil. If bound protein is to be eluted from individual spot positions after probing (Valle at al. 1999; Billich et al. 2002), these should also be marked.
Take the appropriate set of Fmoc-amino acid stock aliquots for cycle 1 from the freezer, bring to room temperature, and activate by adding DIC (4 µl per 100-µl vial; ~0.25 M). Incubate at room temperature for 30 min. For manual experiments, pipette aliquots of the Fmoc-amino acid solutions onto the appropriate spots on the membrane. For automated experiments, place the vials containing the activated Fmoc-AA solutions into the rack of the spotting robot and start cycle 1. Leave for 15 min. Repeat the spotting twice and allow the reaction to proceed for 2 hr (cover the membranes on the spotter with glass plates).
Monitor the amino acid coupling reaction by inspection of the spot color change. Spots should turn yellow-green during this step. If some spots remain dark blue, additional applications of Fmoc-amino acid stock solution can be made.

For the introduction of randomized X positions in the peptide sequences, take the appropriate Fmoc-AA-mix stock solution from the freezer, bring to room temperature, and activate by adding DIC (1.5 µl per 100 µl-vial; ~0.09 M). Incubate at room temperature for 30 min. Perform spotting four times.

Wash each membrane twice with 20 ml of acetylation mix (once for 30 sec, and twice for 2 min). Incubate the membranes in fresh acetylation mix for about 10 min (until all remaining blue color has disappeared).
Wash each membrane in 20 ml of DMF (3 times for 2 min each).
Add 20 ml of piperidine mix and incubate for 5 min.
Wash each membrane in 20 ml of DMF (at least 6 times).
Incubate membranes in 20 ml of DMF containing 1% BPB.

Again, the spots should be stained only light blue. If traces of remaining piperidine on the membranes turn the liquid dark blue, replace the solution and continue the staining. BPB staining is charge-specific. Therefore, it does not only bind to amino-terminal amino groups. The side chains and protecting groups of the amino acids in the peptide chain can strongly influence staining intensity. The visible color of the peptides depends on the overall charge and, therefore, on the individual amino acid sequence.
Wash each membrane with 20 ml of methanol or ethanol (2 times for 5 min).
Transfer the membranes to 3MM paper folders and dry them using cold air from a hair dryer.
Repeat this procedure from steps 2 to 10 for successive elongation cycles.

Stage 4: Terminal Acetylation

Synthetic peptides mimicking fragments of a longer continuous protein chain should be amino-terminally acetylated to avoid the production of an artificially charged terminus. Alternatively, special detection labels can be attached to the amino termini of peptides by spotting respective derivatives. This is useful, for example, when peptides are applied as protease substrates and the enzyme activity is followed through the change of the label upon cleavage of the peptide. We have successfully added biotin via its in situ formed HOBt-ester (normal activation procedure) or fluorescein via its isothiocyanate (FITC) dissolved in DMF.

After the final amino acid elongation cycle from the protocol above, continue as follows:

Incubate each membrane in 20 ml of acetylation mix for a minimum of 30 min (until all remaining blue color has disappeared).
Wash the membranes in 20 ml of DMF (3 times for 2 min), and then in 20 ml of alcohol (2 times for 5 min).
Transfer the membranes to 3MM paper folders and dry them using cold air from a hair dryer.

Stage 5: Side-Chain Deprotection

After the peptide assembly is complete, all side-chain-protecting groups are removed from the peptides. Trifluoroacetic acid is extremely hazardous; the following procedure must be performed in a chemical fume hood!

Prepare 40 ml of deprotection mix.
Place the dried membrane in the reaction trough, add deprotection mix, close the trough very tightly, and incubate overnight with gentle agitation. This harsh treatment is required for complete cleavage of protecting groups (Kramer et al. 1999; Zander 2004). Cellulose membranes less resistant than AC-S will not survive this treatment!)
Subject each membrane to the following series of washes:

20 ml of DCM (4 times for 5 min each)
20 ml of DMF (3 times for 5 min each)
20 ml of alcohol (3 times for 5 min each)
20 ml of acetic acid (1 M in water) (3 times for 5 min each)

This is to remove the Boc group from tryptophan residues.

Wash each membrane with 20 ml of alcohol (3 times).
Transfer the membranes to 3MM paper folders and dry them using cold air from a hair dryer. Store dried membranes in a sealed plastic bag at -20°C, or process further as described in the next section.

Stage 6: Protein-binding Assay

This basic procedure has been optimized for use with AP-conjugated detection antibody and a color signal development. As mentioned above, horseradish peroxidase-labeled detection agents require the use of hydrogen peroxide, which gradually destroys the peptides on the array. More sensitive detection can be achieved with a chemiluminescent substrate of AP (e.g., Immun-Star). If such a substrate is used, follow the manufacturer's instructions for steps 9 to 11 of Method A. Alternatively, test proteins can be labeled prior to incubation with the peptide array by chemical biotinylation and subsequently detected using AP-conjugated streptavidin (under the same conditions as for AP secondary antibody). If fluorescent or radioactive labeled reagents are used, adapt steps 5 to 11 of Method A accordingly. As an easy alternative to chemical labeling, in vitro coupled transcription/translation systems (TNT, Promega) can be recommended (Niebuhr et al. 1997).

Important: If using Method A, prior to probing your protein with the peptide spots on the membrane, always do a "pre-run" in which you first apply this protocol while omitting steps 5 and 6. This is necessary to control for unspecific signals from components of the detection process or remaining proteins from a previous experiment on the same membrane. However, in case the proteins are electro-transferred and detected on a secondary nitrocellulose membrane (Method B, below), this precaution does not apply.

Method A

Place a single membrane in a polystyrene plate, and wet it with a few drops of methanol or ethanol.

This is to enhance rehydration of any peptide spots that might be hydrophobic. The peptide locations should not be visible as white spots! If this happens, extend the alcohol treatment in a sonication bath at room temperature until spots have disappeared
Wash the membrane in 10 ml of TBS (3 times for 10 min each).
Incubate overnight in 10 ml of MBS.

The blocking conditions can be critical to the success of an experiment, and, depending on the protein of interest, it may be necessary to try a number of different blocking solutions to optimize the signal-to-noise ratio. The following solutions represent increasingly stringent blocking conditions: (i) 2% (w/v) skim milk powder in TBS, (ii) 2% (w/v) skim milk powder, 0.2% (v/v) Tween-20 in TBS, (iii) MBS, (iv) MBS with 50% (v/v) horse serum. In our hands, blocking solution iii works best for most applications.
Wash the membrane once in 10 ml of T-TBS for 10 min
Incubate for 2-4 hr in the presence of probe antibody (or protein) diluted in 8-10 ml of MBS.

For monoclonal antibodies, or pure proteins, use ~4-5 µg of purified antibody per milliliter of incubation volume. When using a polyclonal serum, we recommend a dilution of 1:100. It is not necessary to use a large volume of protein solution for the incubation. However, make sure that the membrane is completely covered throughout the incubation. To prevent drying out, use a lid, or seal the membrane in a plastic bag.
Wash the membrane 3 times in 10 ml of T-TBS (for 10 min each).
Incubate for 1-2 hr with AP-conjugated secondary antibody, diluted in 10 ml of MBS.
Wash the membrane 2 times in 10 ml of T-TBS (for 10 min each).
Wash the membrane 2 times in 10 ml of CBS (for 10 min each).
Transfer the membrane to a flat glass tray and add 10 ml of CDS. Incubate without agitation until good signals are obtained.

For individual peptides on spots, this usually takes 10-30 min; peptide pools may require longer incubations (2 hr to overnight).
Stop the reaction by washing the membrane twice in PBS. Keep the membrane wet, either in PBS or covered in plastic wrap. Store at 4°C.

The picture of signals on the membrane can now be documented by photography or (better) be electronically digitized with a scanner. A high-quality electronic image can be used to quantify signal intensities. Avoid drying of the membrane at this stage. If the membrane dries out, proteins may denature and become difficult to remove. After successful documentation of signals by photography or electronic scanning, continue with membrane stripping (see below, after Method B).

Method B

If only weak binding of the test protein is anticipated, or if excessive background was observed in Method A, it may help to electro-transfer the bound test protein onto a secondary nitrocellulose membrane (e.g., Protan Nitrocellulose Transfer Membrane from Schleicher & Schuell) and repeat the detection procedure. Here, any detection system appropriate for nitrocellulose can be used (e.g., HRP conjugates), as the peptides will not be affected. Proceed through steps 1-6 of Method A, then continue as follows:

Briefly equilibrate the peptide membrane and a sheet of nitrocellulose, trimmed to fit the peptide membrane, in transfer buffer.
Electro-transfer the proteins bound to the peptide spot membranes onto the nitrocellulose membrane for 1 hr at 0.85 mA/cm2. IMPORTANT: Due to SDS denaturation, all proteins will be negatively charged. Therefore, the nitrocellulose should be placed toward the positive electrode.

Depending on the chemical properties of the protein ligands, the time required for the transfer might differ and, therefore, must be determined empirically.
Block the nitrocellulose membrane with MBS for 2 hr at room temperature.
Incubate the nitrocellulose membrane for 75 min with an AP- or HRP-conjugated detection antibody, or AP-/HRP-streptavidin for biotinylated proteins diluted in MBS.

Use dilutions comparable to those employed in immunoblots after SDS-PAGE.
Wash the nitrocellulose membranes 3 times for 5-10 min each in T-TBS, and then 3 times for 5-10 min in TBS for 5-10 min each.
Remove excess buffer from the nitrocellulose membrane by gently placing tissue onto it.

To avoid damage to the adsorbed protein, do not wipe or press tissue onto the membrane.
Detect the spots using a chemiluminescence detection kit (e.g., ECL western blotting detection reagents from Amersham Biosciences) according to the manufacturer's instructions. Be sure to include a positive control for the kit.

If no signal has been detected after 30 min of exposure, check that the positive control has worked, and repeat the experiment using less stringent blocking. If problems persist, this may indicate a discontinuous binding site or very low affinity binding.

In case of nonspecific signals or high background, increase the stringency of the blocking conditions and make sure that the primary binding partner and detection reagent (e.g., antibody) are of high purity and are used in the highest possible dilution.

Stage 7: Membrane Stripping

A peptide spot membrane that was used in a protein-binding assay can be stripped off bound protein and reused for multiple-protein binding assays. In principle, thanks to the stability of the immobilized peptides, membranes can be regenerated up to 50 times without loss of signal intensity.

In some cases, proteins can resist removal from the spots, and those membranes can be used once only for Method A (on-spot membrane detection). Remains of bound protein must be checked by probing a stripped spot membrane first with the detection system (see above, Stage 6, Protein-binding Assay protocol). Alternatively, Method B of Stage 6 can be applied.

Use the following procedure to strip the membrane.

Wash the spot membrane 2 times for 10 min in 20 ml of water.
Incubate in 20 ml of DMF, until the blue color of spot signals has dissolved (usually about 10 min; incubate in a sonication bath at 40°C if necessary). Remove the solution and wash once more for 10 min in 20 ml of DMF for 10 min.

This step can be omitted if a detection method other than a dye precipitation or Method B of Stage 6 was used.
Subject the membrane to the following series of washes:

20 ml of water (3 times for 10 min each)
20 ml of SM-A in a sonication bath at 40°C (3 times for 10 min each)
20 ml of SM-B (3 times for 10 min each)
20 ml of alcohol (3 times for 10 min each)
Start the new binding assay at step 2 of the protein-binding assay protocol of Stage 6 or transfer the membranes to 3MM paper folders and dry them using cold air from a hair dryer. Store dried membranes in a sealed plastic bag at -20°C.

SUMMARY

These protocols aim to help researchers, including non-chemists, to prepare high-quality, low-cost, synthetic peptide arrays for a variety of biological screening experiments, prominently for the detailed molecular study of protein-protein interactions. These protocols are tried and tested. They even work successfully under extreme conditions such as the tropical heat of an Argentinian summer, with lab temperatures of about 35°C, with the dichloromethane almost boiling and humidity over 90%. The authors are happy to give advice in case of problems with the procedures, or to suggest adaptations that might be useful for other applications.

REFERENCES

Billich C., Sauder, C., Frank R., Herzog S., Bechter K., Takahashi K., Peters H., Staeheli P., and Schwemmle M. 2002. High-avidity human serum antibodies recognizing linear epitopes of Borna disease virus proteins. Biol. Psychiatry 51 : 979-987.

Kramer A., Reineke U.,Dong L., Hoffmann B., Hoffmuller U., Winkler D., Volkmer-Engert R., and Schneider-Mergener J. 1999. Spot synthesis: Observations and optimizations. J. Pept. Res . 54 : 319-327.

Niebuhr K., Ebel F., Frank R., Reinhard M., Domann E., Carl U.D., Walter U., Gertler F.B., Wehland J., and Chakraborty T. 1997. A novel proline-rich motif present in the ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family. EMBO J . 17 : 5433-5444.

Valle M., Kremer L., Martinez C., Roncal F., Valpuesta J.M., Albar J.P., and Carrascosa J.L. 1999. Domain architecture of the bacteriolphage 29 connector protein. J. Mol. Biol . 288 : 899-909.

Zander N. 2004. New planar substrates for the in situ synthesis of peptide arrays. Mol Divers . 8 : 189-195.

Anyone using the procedures in this protocol does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in this protocol and has no liability in connection with the use of these materials. Materials used in this protocol may be considered hazardous and should be used with caution. For a full listing of cautions regarding these material, please consult:
Protein: Protein Interactions, Second Edition: A Molecular Cloning Manual , edited by Erica A. Golemis and Peter D. Adams, © 2005 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 591.