Protein microarray technology

Abstract

Protein chips have emerged as a promising approach for a wide variety of applications including the identification of protein-protein interactions, protein-phospholipid interactions, small molecule targets, and substrates of proteins kinases. They can also be used for clinical diagnostics and monitoring disease states. This article reviews current methods in the generation and applications of protein microarrays.

Fig. 1

Applications of functional protein microarrays. A representative sample of the different assays that have been performed on functional protein microarrays. Proteins are immobilized at high spatial density onto a microscope slide and the slide can then be probed for various interactions. While Cy5 is the fluorophore shown, many other fluorophores can be used for detection.

Fig. 2

Protein attachment methods. (A) Proteins can be attached randomly via different chemistries including aldehyde- and epoxy-treated slides that covalently attach protein by their primary amines or by adsorpition onto slides coated with nitrocellulose or acrylamide gel pads. (B) Proteins can be uniformly orientated onto slides coated with a ligand. For example, His6X-tagged proteins can be bound to nickel-derivatized slides and biotinylated proteins can be attached to streptavidin-coated slides. This leads to attachment through the tag and presumably orientates the protein away from the slide surface.

Fig. 3

Identification of DNA-binding proteins using a functional protein microarray. Genomic DNA is purified, fragmented, and labeled with Cy3-dCTP. A yeast proteome array with the majority of the yeast proteins was incubated with the labeled DNA to identify novel DNA-binding proteins, including Arg5,6, a mitochondrial enzyme.

Fig. 4

(A) A yeast proteome microarray containing ∼4400 GST-tagged yeast proteins printed in duplicate. The slide was probed with anti-GST antibodies followed by Cy5-labeled anti-rabbit antibodies. Forty of 48 blocks are shown. (B) A kinase assay with ³³P-γ-ATP and active kinase on a yeast proteome microarray. Dark spots represent radiolabeled phosphorylated substrates on the array. Kinases that autophosphorylate are printed in each of the 40 blocks (shown above in blue boxes) and serve as reference points on the slide.