Biopolymers & Proteomics Laboratory

The David H.Koch Institute for Integrative Cancer Research at MIT

Bldg 76 Room 181

Telephone: 617-253-7038

Cellulose SPOT Synthesis Peptide Array (Membrane)


SPOT synthesis allows screening of synthetic peptides or other compounds arrayed in 20 x 30 formation on a 10 x 15 cm cellulose membrane. Sequences of spots are usually emailed to the lab as text, spreadsheet or database documents. Instrument software allows us to create the spot sequences and residue offsets from an intact protein sequence. Alanine scanning, truncation or replacement sequences, for example, can also be generated by the software from a single peptide sequence thus eliminating the need for researchers to create a list of 600 sequences although you can certainly do that if desired. See the Example Arrays section for more information.

Description of Intavis SPOT Synthesis Peptide Arrayer

The Intavis SPOT synthesis peptide arrayer system consists of a computer controlled Gilson, Inc. diluter and XYZ liquid handling robot which allows the deposition on amino-PEG cellulose membranes of individual activated amino acids resulting in peptide formation using FMOC chemistry. The practical upper limit is about 40 residues in length although 30-25 residues is a more reasonable upper limit. The shorter the epitope, the more flawless the spot product.

Two membranes, which can be probed and stripped perhaps 5-10 times, can be synthesized simultaneously. Each membrane allows up to 600 peptide spots. Each spot consists of approximately 40 nmol starting peptide. Basically, activated FMOC residues are deposited in a 200 nanoliter volume forming a spot with a diameter of approximately 2-3 mm. After coupling, the membrane is acetylated to prevent unwanted synthesis between the spots.

The FMOC group is then removed and synthesis of the desired peptide continues. Activation occurs via a DIC/HOBT strategy. De-blocking, washing, acetylation and final side-chain deprotection steps are performed automatically. Four residues can be coupled per day. The membranes are gridded with pencil and you are provided with a printout and/or file that details which sequences are contained on each spot.

Up to 44 amino acid reservoirs are available allowing for the synthesis of, for example, a membrane containing 20 L-form residues, 20 D-form amino acids plus four nonstandard residues such as phosphor Ser/Thr/Tyr and biotin. If your experiment requires fewer than 600 spots you may wish to make a copy of the array on the same membrane, perhaps with a different offset, to verify hits or to cut the membrane and allow additional experiments.


References: (more probing and stripping refs available upon request)

Analysis of Protein Interactions with Immobilized Peptide Arrays Synthesized on Membrane Supports (2006). This protocol was adapted from “Analysis of Protein Interactions with Immobilized Peptide Arrays Synthesized on Membrane Supports,” contributed by Ronald Frank and Stefan Dübel, Chapter 31, in Protein-Protein Interactions, 2nd edition (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

Tetrahedron, Vol. 48, No. 42, pp 9217-9232, 1992, Ronald Frank, "Spot synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support."

Tetrahedron Letters, Vol. 31, No. 40, pp 5811-5814, 1990, Andrew Bray, N. Joe Maeji and H. Mario Geysen, "The simultaneous multiple production of solution phase peptides; assessment of the Geysen Method of simultaneous peptide synthesis."

Applications for SPOT Synthesis Peptide Arrays

Screening format, molecular recognition of immobilized peptides
Antibody epitope mapping
Amino acid substitution or truncation study of epitopes
Receptor/ligand interactions
Metal binding studies
Protein/Peptide; Protein/Protein interactions
Identification of important residues- amino acid replacement studies
Nucleic acid/Protein interaction
Enzymatic activity  (kinase and phosphatase, enzyme substrate )
High resolution characterization of binding motifs
Assays requiring free peptides - Cleavable and light sensitive linkers can be coupled to allow solution phase peptides

Quality Control

We request that you leave us 1-3 three spots to make test peptides which we analyze by mass spectrometry.
You may also want to print an epitope tag alanine scan and probe it with antibody. For example:

  • HA Tag: YPYDVPDYA influenza hemagglutinin epitope monoclonal antibody HA.11
  • N-Myc Tag: SPYVESEDAPPQKC (aa 327-339) of human N-myc.

Example: alanine scan of the HA tag:

Example Arrays

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