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ANALYTICAL CURRENTS Self-assembling protein arrays Although protein microarrays are potentially useful for high-throughput screens of protein interactions, they have not been widely adopted. Current methods of fabricating protein microarrays involve purifying mammalian proteins, often from yeast or bacteria, then spotting the proteins onto a slide. But purifying mammalian proteins is difficult, and some proteins are not stable for long periods of time on an array. Joshua LaBaer and colleagues at Harvard Medical School have developed a new way of producing protein arrays that overcomes these limitations. Instead of purifying mammalian proteins from yeast or bacteria, LaBaer and colleagues make proteins directly on a microarray slide. Complementary DNAs (cDNAs) encoding for various mammalian proteins are adhered onto a glass slide via biotin/avidin linkages at different spots. When the researchers are ready to use the nucleic acid programmable protein array (NAPPA), a commonly used cell-free mammalian lysate is added to the entire slide surface. The lysate transcribes the cDNA into mRNA and translates the
Target DNA
Target protein
GST antibody
Avidin Add cell-free expression system A schematic of the NAPPA approach for producing protein arrays. (Adapted with permission. Copyright 2004 American Association for the Advancement of Science.)
mRNA into protein. Each protein is fused to a glutathione-S-transferase (GST) tag and then captured and held onto the slide by a GST antibody that was spotted next to the cDNA. In every spot, ~10 fmol of protein were produced and immobilized, an amount comparable to other array methods. To test the NAPPA method, the researchers bound eight different target proteins onto the array. In separate experiments, two query proteins tagged with the hemagglutinin (HA) epitope were assayed for interaction with the eight targets. Interactions were detected by probing the array for the HA tag. Each query protein interacted specifical-
ly with its known interacting proteins on the array. In a larger experiment, previously known and unknown interactions among 29 different proteins involved in human DNA replication were identified. A few technical hurdles must still be overcome for the NAPPA method to detect certain binding events. For example, the presence of a third protein may be necessary to bridge an interaction. Appropriate posttranslational modifications, which may be necessary for particular interactions, may not be present after the proteins are translated in the NAPPA method. (Science 2004, 305, 86–90)
Improved surface plasmon fluorescence Although surface plasmon fluorescence
cules as a scaffold on which the target
protein sample is added and tested for in-
spectroscopy allows researchers to moni-
proteins reside. This approach moves the
teraction with the target protein. Using
tor binding kinetics directly, the thin metal
protein interaction away from the surface
mouse IgG as the target protein and fluo-
layer on a surface plasmon resonance
and enables full fluorescence detection.
rescently labeled antimouse antibodies as
(SPR) chip can quench the fluorescence
The researchers start with commercial-
the analyte, Knoll and co-workers deter-
signal when a fluorophore is near the sur-
ly available sensor chips in which 100-nm-
mined a detection limit of 500 aM. A com-
face. To overcome this limitation, Wolfgang
long carboxymethyldextran (CMD) chains
bination of SPR and fluorescence data re-
Knoll and co-workers at the Max Planck
are tethered via a self-assembled mono-
vealed that ~10 antibody molecules bound
Institute for Polymer Research (Germany)
layer. The target protein is attached to the
every minute. (J. Am. Chem. Soc. 2004, 126,
and Biacore AB (Sweden) use long mole-
CMD matrix by a covalent bond. Next, a
8902–8903)
© 2004 AMERICAN CHEMICAL SOCIETY
S E P T E M B E R 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y
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