The Panorama Human p53 Protein Functional Microarray contains 49 p53
SNP variants and one wild-type control. This array utilizes a proprietary
biotin-carboxy carrier protein-tagging technology that ensures only
functional proteins are printed on the microarray slides. The protein
sequences are derived from the Mammalian Gene Collection (MGC), expressed
in insect cells, and analyzed to verify protein sequence.
Background
Figure 1. Arrayed proteins are
captured onto glass slides via the BCCP tag. The tag ensures that
the immobilized proteins are oriented for possible probe
interactions, strongly bound via a biotin:streptavidin linkage,
and easily purified and captured in a single step.
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The microarray utilizes a proprietary tagging technology based on the
presence and biotinylation of a biotin-carboxy carrier protein (BCCP) tag
to ensure only correctly folded and fully functional proteins are
immobilized on a streptavidin-coated slide.
Figure 2. Functional p53 protein
microarrays were probed with Cy3-GADD45 DNA that bound to a subset
of arrayed p53 proteins (A). After the DNA-binding assay was
performed, the presence and amounts of p53 variant proteins
spotted were confirmed using a Cy5-anti-p53 antibody (B).
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The tagging system utilizes a 50-Å spacer arm, thereby allowing proteins
to be presented in a similar manner and maximizing the opportunity for
active sites to interact with binding partners (Figure 1). After
expression, western blots are performed to verify full-length proteins. In
addition, the clones encoding the proteins are fully sequence verified to
ensure the expressed proteins arrayed are of the correct sequence.
To demonstrate the utility of this technology, functional p53
microarrays were probed with a Cy3-GADD45 oligonucleotide representing the
GADD45 DNA-promoter element. GADD45 binds to wild-type p53 and will
differentially bind p53 variants. After the DNA-binding assay was
performed (Figure 2), the presence and amounts of the p53 variant proteins
were confirmed using a Cy5-anti-p53 antibody (Figure 3).
Figure 3. Relative DNA-probe binding
and p53 conformation is independent of antibody binding to p53
mutants. Data (RFU) from figure 3 were normalized to wild-type
p53. Some p53 mutations affected the binding of Cy3-GADD45 (blue
bar), while others demonstrated little or no change in DNA binding
activity (e.g., P82L). Decreases (e.g., M113T) and increases
(e.g., R337C) in DNA binding were observed. Subsequent to the
DNA-binding assay, incubation with a Cy5-anti- p53 antibody (pink
bar) was performed on the array to confirm the relative amounts of
each spotted p53 protein.
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