Cell-free protein array: Difference between revisions

Revision as of 22:24, 19 March 2008

Revision as of 22:26, 19 March 2008

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[[Image:Figure_3_puromycin2.png|thumb|600px|'''Figure 3: Schematic 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 hybrizedhybridized 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> (Figure 3). 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===