Cite as: Cold Spring Harb. Protoc.; 2008; doi:10.1101/pdb.prot5061

This Protocol
Right arrow Full Text
Right arrow Update/discuss this protocolDiscussion icon
Right arrow Alert me when this protocol is cited
Right arrow Alert me when comments are published
Right arrow Alert me if a correction is posted
Services
Right arrow Similar protocols in this database
Right arrow Alert me to new releases of protocols
Right arrow Save to Personal Folders
Right arrow Download to citation manager
Right arrow Printer-friendly versionPrinter-friendly version
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Link, A. J.
Right arrow Articles by LaBaer, J.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Link, A. J.
Right arrow Articles by LaBaer, J.
Related Collections
Right arrow Antibodies, general
Right arrow CSHL Proteomics Course
Right arrow Protein Identification and Analysis
Right arrow Visualization
Right arrow Visualization of Proteins
Right arrow High-Throughput Analysis, general
Right arrow Molecular Biology, general
Right arrow cDNA
Right arrow Proteins and Proteomics, general
Right arrow Immunodetection
Right arrow Proteomics
Right arrow Microarrays
Right arrow Microarrays, general
Right arrow Spotted Glass Microarrays
Right arrow Proteome Analysis
Right arrow Expression Libraries
Right arrowRelated Protocols
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?
BSN globe

protocolProtocol

Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 6: Detecting Proteins 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

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


INTRODUCTION

Functional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. This method uses cell-free extracts that transcribe and translate DNA into proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target proteins. Instead of printing proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of proteins and DNA using antibodies and stains. This protocol describes antibody detection of the arrayed proteins.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?

Related Protocols

Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 1: Coating Glass Slides with Amino Silane
Andrew J. Link and Joshua LaBaer
CSH Protocols 2008: 5056. [Abstract] [Full Text]

Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 2: Preparing Bacterial Cultures in a 96-Well Format
Andrew J. Link and Joshua LaBaer
CSH Protocols 2008: 5057. [Abstract] [Full Text]

Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 3: Isolating DNA Plasmids in a 96-Well Plate Format
Andrew J. Link and Joshua LaBaer
CSH Protocols 2008: 5058. [Abstract] [Full Text]

Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 4: DNA Biotinylation, Precipitation, and Arraying of Samples
Andrew J. Link and Joshua LaBaer
CSH Protocols 2008: 5059. [Abstract] [Full Text]

Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 5: Expressing Proteins on NAPPA Slides
Andrew J. Link and Joshua LaBaer
CSH Protocols 2008: 5060. [Abstract] [Full Text]

Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 7: Detecting DNA on NAPPA Slides
Andrew J. Link and Joshua LaBaer
CSH Protocols 2008: 5062. [Abstract] [Full Text]



This article has been cited by other articles:


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

Home page
 CSH ProtocolsHome page
A. J. Link and J. LaBaer
Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein-Protein Interactions. Protocol 1: Coexpression of Query Protein on NAPPA Slides
CSH Protocols, December 1, 2008; 2008(13): pdb.prot5108 - pdb.prot5108.
[Abstract] [Full Text]

Home page
 CSH ProtocolsHome 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
CSH Protocols, December 1, 2008; 2008(13): pdb.prot5109 - pdb.prot5109.
[Abstract] [Full Text]

Home page
 CSH ProtocolsHome page
A. J. Link and J. LaBaer
Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 1: Coating Glass Slides with Amino Silane
CSH Protocols, November 1, 2008; 2008(12): pdb.prot5056 - pdb.prot5056.
[Abstract] [Full Text]

Home page
 CSH ProtocolsHome page
A. J. Link and J. LaBaer
Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 2: Preparing Bacterial Cultures in a 96-Well Format
CSH Protocols, November 1, 2008; 2008(12): pdb.prot5057 - pdb.prot5057.
[Abstract] [Full Text]

Home page
 CSH ProtocolsHome page
A. J. Link and J. LaBaer
Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 3: Isolating DNA Plasmids in a 96-Well Plate Format
CSH Protocols, November 1, 2008; 2008(12): pdb.prot5058 - pdb.prot5058.
[Abstract] [Full Text]

Home page
 CSH ProtocolsHome page
A. J. Link and J. LaBaer
Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 4: DNA Biotinylation, Precipitation, and Arraying of Samples
CSH Protocols, November 1, 2008; 2008(12): pdb.prot5059 - pdb.prot5059.
[Abstract] [Full Text]

Home page
 CSH ProtocolsHome page
A. J. Link and J. LaBaer
Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 5: Expressing Proteins on NAPPA Slides
CSH Protocols, November 1, 2008; 2008(12): pdb.prot5060 - pdb.prot5060.
[Abstract] [Full Text]

Home page
 CSH ProtocolsHome page
A. J. Link and J. LaBaer
Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 7: Detecting DNA on NAPPA Slides
CSH Protocols, November 1, 2008; 2008(12): pdb.prot5062 - pdb.prot5062.
[Abstract] [Full Text]