Cell-free protein array: Difference between revisions

Revision as of 16:37, 17 July 2013

Revision as of 22:41, 10 September 2013

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====Protein ''in situ'' array (PISA)====

Unlike NAPPA, PISA<ref>He, M. and M. J. Taussig (2001). "Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method)." Nucleic Acids Res 29(15): E73-3.</ref> completely bypasses DNA immobilization as the DNA template is added as a free molecule in the reaction mixture. In 2006, another group refined and miniaturized this method by using multiple spotting technique to spot the DNA template and cell-free transcription and translation mixture on a high-density protein microarray with up to 13,000 spots.<ref>Angenendt, P., J. Kreutzberger, et al. (2006). "Generation of high density protein microarrays by cell-free in situ expression of unpurified PCR products." Mol Cell Proteomics 5(9): 1658–66.</ref>. This was made possible by the automated system used to accurately and sequentially supply the reagents for the transcription/translation reaction occurs in a small, sub-nanolitre droplet.

[[Image:Figure 3 puromycin2.png|thumb|600px|'''Diagram of ''In situ'' puromycin-capture''']]

====''In situ'' puromycin-capture====

This method is an adaptation of [[mRNA display]] technology. [[PCR]] DNA is first transcribed to [[mRNA]], and a single-stranded DNA [[oligonucleotide]] modified with [[biotin]] and [[puromycin]] on each end is then hybridized to the 3’-end of the mRNA. The mRNAs are then arrayed on a slide and immobilized by the binding of biotin to [[streptavidin]] that is pre-coated on the slide. Cell extract is then dispensed on the slide for ''in situ'' translation to take place. When the ribosome reaches the hybridized oligonucleotide, it stalls and incorporates the puromycin molecule to the nascent [[polypeptide]] chain, thereby attaching the newly synthesized protein to the microarray via the DNA oligonucleotide.<ref>Tao, S. C. and H. Zhu (2006). "Protein chip fabrication by capture of nascent polypeptides." Nat Biotechnol 24(10): 1253–4.</ref>. A pure protein array is obtained after the mRNA is digested with [[RNase]]. The protein spots generated by this method are very sharply defined and can be produced at a high density.

===Nano-well array format===

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==Applications==

Protein interactions: To screen for [[protein–protein interactions]]<ref name="Ramachandran, N. 2004"/> and protein interactions with other molecules such as [[metabolite]]s, [[lipid]]s, DNA and small molecules.;<ref>He, M. and M. W. Wang (2007). "Arraying proteins by cell-free synthesis." Biomol Eng 24(4): 375–80.</ref>; enzyme inhibition assay:<ref name="Angenendt, P. 2004"/> for high throughput drug candidate screening and to discover novel [[enzyme]]s for use in [[biotechnology]]; screening antibody specificity.<ref>He, M. and M. J. Taussig (2003). "DiscernArray technology: a cell-free method for the generation of protein arrays from PCR DNA." J Immunol Methods 274(1–2): 265–70.</ref>

==References==

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==External links==

*[http://biodesign.asu.edu/labs/labaer/research NAPPA]

*[http://www.discerna.co.uk/discerna_discerna_technologies_arrays.htm PISA and DAPA]{{dead link|date=July 2013}}

*[http://www.functionalgenomics.org.uk/sections/resources/protein_arrays.htm Protein arrays resource page]