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Assessing biofilm inhibition and immunomodulatory activity of small amounts of synthetic host defense peptides synthesized using SPOT-array technology


Peptides are promising drug candidates because of their diversity, biocompatibility and spectrum of activities. Here, we describe a protocol for high-throughput screening of SPOT-peptide arrays to assess the antibiofilm, antimicrobial and immunomodulatory activities of synthetic peptides. It is a Protocol Extension of our previous Nature Protocols article, which describes the synthesis of SPOT-peptide arrays and assays for screening antimicrobial activity. This latest protocol allows the simultaneous assessment of hundreds of synthetic host defense peptides to define their overall activity profiles and identify candidate sequences that are suitable for further characterization and development as anti-infectives. When coupled with the SPOT-array technology for peptide synthesis, the described procedures are rapid, inexpensive and straightforward for peptide library screening. The protocols can be implemented in most microbiology or immunology research laboratories without the need for specialists. The time to complete each step ranges between 1 and 4 h with overnight pauses, and datasets related to the antibiofilm and immunomodulatory activities of a large set of peptide sequences can be generated in a few days.

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Fig. 1: Diagram of the steps of the biofilm inhibition screening assay.
Fig. 2: Schematic diagram of the experimental setup to screen the immunomodulatory activity of the SPOT peptides.
Fig. 3: Biofilm inhibition activities of 96 different single amino acid substitution variants of the HDP 1018 synthesized on a SPOT array.
Fig. 4: Example results from the screening of SPOT peptides for their immunomodulatory activity.

Data availability

Data presented in this protocol (dataset for Figs. 3 and 4) are available in the supporting primary research articles (refs. 26,27), and raw data are available from the corresponding author upon request.


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We acknowledge the financial assistance of the Canadian Institutes of Health Research, Foundation grant FDN-154287 to R.E.W.H. for funding peptide research in our laboratory. H.E. is the recipient of a UBC Killam Fellowship and a Research Trainee Award from the Michael Smith Foundation for Health Research. R.E.W.H. is a Canada Research Chair in Health and Genomics and holds a UBC Killam Professorship.

Author information




H.E. and E.F.H. wrote the protocol. H.E. drew the illustrations in Figs. 1 and 2. R.E.W.H. extensively edited the article.

Corresponding author

Correspondence to Robert E. W. Hancock.

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Competing interests

E.F.H. and R.E.W.H. have filed patents related to the antibiofilm and immunomodulatory functions of synthetic HDPs. These patents have been assigned to their employer, the University of British Columbia, and licensed to ABT Innovations Inc., in which R.E.W.H. has an ownership position.

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Peer review information Nature Protocols thanks Donald Davidson, Sam Walker and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Haney, E. F. et al. Sci. Rep. 8, 1871 (2018):

Haney, E. F., Mansour, S. C., Hilchie, A. L., de la Fuente-Nunez, C. & Hancock, R. E. W. Peptides 71, 276–285 (2015):

Haney, E. F., Barbosa, S. C., Baquir, B. & Hancock, R. E. W. J. Med. Chem. 62, 10294–10304 (2019):

This protocol is an extension to: Nat. Protoc. 2, 1333–1349 (2007):

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Etayash, H., Haney, E.F. & Hancock, R.E.W. Assessing biofilm inhibition and immunomodulatory activity of small amounts of synthetic host defense peptides synthesized using SPOT-array technology. Nat Protoc 16, 1850–1870 (2021).

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