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{{Short description|DNA technology}}
'''Cell-free protein array''' technology produces [[protein microarray]]s by performing [[in vitro]] synthesis of the target proteins from their [[DNA]] templates. This method of synthesizing protein microarrays overcomes the many obstacles and challenges faced by traditional methods of protein array production<ref name="Stevens, R. C. 2000">Stevens, R. C. (2000). "Design of high-throughput methods of protein production for structural biology." Structure 8(9): R177-R185.</ref> that have prevented widespread adoption of protein microarrays in [[proteomics]]. Protein arrays made from this technology can be used for testing [[protein–protein interactions]], as well as protein interactions with other cellular molecules such as DNA and lipids. Other applications include enzymatic inhibition assays and screenings of antibody specificity.▼
▲'''Cell-free protein array''' technology produces [[protein microarray]]s by performing ''[[in vitro]]'' synthesis of the target proteins from their [[DNA]] templates. This method of synthesizing protein microarrays overcomes the many obstacles and challenges faced by traditional methods of protein array production<ref name="Stevens, R. C. 2000">Stevens, R. C. (2000). "Design of high-throughput methods of protein production for structural biology." Structure 8(9): R177-R185.</ref> that have prevented widespread adoption of protein microarrays in [[proteomics]]. Protein arrays made from this technology can be used for testing [[protein–protein interactions]], as well as protein interactions with other cellular molecules such as DNA and lipids. Other applications include enzymatic inhibition assays and screenings of antibody specificity.
==Overview / background==▼
The runaway success of [[DNA microarray]]s has generated much enthusiasm for protein microarrays. However, protein microarrays have not quite taken off as expected, even with the necessary tools and know-how from DNA microarrays being in place and ready for adaptation. One major reason is that protein microarrays are much more laborious and technically challenging to construct than DNA microarrays.
The traditional methods of producing protein arrays require the separate ''[[in vivo]]'' expression of hundreds or thousands of proteins, followed by separate purification and immobilization of the proteins on a solid surface. Cell-free protein array technology attempts to simplify protein microarray construction by bypassing the need to express the proteins in
The target proteins are synthesized ''[[in situ]]'' on the protein microarray, directly from the DNA template, thus skipping many of the steps in traditional protein microarray production and their accompanying technical limitations. More importantly, the expression of the proteins can be done in parallel, meaning all the proteins can be expressed together in a single reaction. This ability to multiplex protein expression is a major time-saver in the production process.
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====''In situ'' puromycin-capture====
This method is an adaptation of [[mRNA display]] technology. [[Polymerase chain reaction|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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==References==
<ref name="Protein arrays: recent achievements and their application to study the human proteome.">
{{reflist}}
==External links==
*[https://web.archive.org/web/20090928065617/http://www.biodesign.asu.edu
*[https://web.archive.org/web/20090105152214/http://www.discerna.co.uk/discerna_discerna_technologies_arrays.htm PISA and DAPA]
*[https://web.archive.org/web/20080220114252/http://www.functionalgenomics.org.uk/sections/resources/protein_arrays.htm Protein arrays resource page]
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