| 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. |
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| 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)
Acetic acid
DMF
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)
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
Piperidine mix
Methanol or ethanol, technical grade (95%)
Deprotection mix
Trifluoroacetic acid is very harmful and volatile, and should be handled with gloves under a hood!
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
Tris-buffered saline (TBS)
T-TBS: TBS buffer plus 0.05% Tween-20
Phosphate-buffered saline (PBS)
Citrate-buffered saline (CBS)
Membrane blocking solution (MBS)
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)
Stripping mix A (SM-A)
Stripping mix B (SM-B)
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| Special Equipment |
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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
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| 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. |
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Stage 1: Preparative WorkGenerate 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.
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Stage 2: Generation of the SPOT ArrayMark 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.
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Stage 3: Assembly of Peptides on SPOTsTake 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.
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