Cite as: Cold Spring Harb. Protoc.; 2008; doi:10.1101/pdb.prot5108

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Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein-Protein Interactions. Protocol 1: Coexpression of Query Protein on NAPPA Slides

Andrew J. Link1 and Joshua LaBaer2,3

1 Vanderbilt University School of Medicine, Nashville, TN 37232-8575, USA
2 Harvard University School of Medicine, Harvard Institute of Proteomics, Cambridge, MA 02141-2023, USA

3Corresponding author (joshua_labaer{at}hms.harvard.edu)


INTRODUCTION

The Nucleic Acid Programmable Protein Array (NAPPA) approach for producing protein microarrays uses cell-free extracts to transcribe and translate cDNAs encoding target proteins directly onto glass slides. Following array preparation, interactions with a protein of interest (query protein) are detected either by probing an expressed NAPPA slide with the purified query protein or by coexpressing the query protein on the NAPPA slide at the same time that the target proteins are expressed. This protocol describes the coexpression method, which involves adding the gene for the query protein to the cell-free protein expression mix. The amount of query protein that is transcribed and translated from the corresponding plasmid DNA depends on the amount of plasmid DNA used and the size of the protein of interest, among other factors. If too little query protein is expressed, there may be no detectable binding signal. Excessive amounts of protein expression may generate nonspecific background signals. Because the optimum amount of query plasmid varies with each query protein, it is essential to assess empirically the optimal amount of query protein DNA to add to a coexpression experiment.


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Related Article

Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein-Protein Interactions: General Guidelines
Andrew J. Link and Joshua LaBaer
Cold Spring Harb Protoc 2008: 62. [Abstract] [Full Text]

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Home page
 Cold Spring Harb ProtocHome page
A. J. Link and J. LaBaer
Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein-Protein Interactions: General Guidelines
Cold Spring Harb Protoc, December 1, 2008; 2008(13): pdb.ip62 - pdb.ip62.
[Abstract] [Full Text]

Home page
 Cold Spring Harb ProtocHome page
A. J. Link and J. LaBaer
Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein-Protein Interactions. Protocol 2: Detection of Query Proteins on NAPPA Slides
Cold Spring Harb Protoc, December 1, 2008; 2008(13): pdb.prot5109 - pdb.prot5109.
[Abstract] [Full Text]