THE HUMAN PROTEOME ARRAY: HuProt™
HuProt™ — The Human Proteome Microarray
>21,000 GST-purified recombinant proteins. Expressed in yeast from sequence-confirmed plasmids. Piezoelectrically printed with duplicate spots. Confirmed folding by kinase autophosphorylation assay. Batches of up to 1000 slides.
There have been other protein microarrays, but none were made from a protein library as comprehensive or thoroughly validated as HuProt. HuProt arrays allow for pan-isotype autoantibody biomarker profiling versus properly folded, three-dimensional human proteins.
HuProt™ — Content Library
The new HuProt v4.0 consists of >21,000 unique human proteins, isoform variants, and protein fragments – covering 16,794 unique genes. This includes 15,889 of the 19,613 canonical human proteins described in the Human Protein Atlas, with broad coverage across protein subclasses:
HuProt™ — Production and QA/QC
Each HuProt array begins as CDI’s ORF collection, which we mobilize into a yeast galactose-inducible GST fusion vector (pEGH-A) using Gateway-mediated site-specific recombination. Each of our >21,000 clones is individually verified to have correct inserts by both BsrGI digestion and plasmid DNA sequencing. Clones with confirmed size, sequence, and high copy number are preserved in our library, which is routinely transformed into yeast for large-scale protein expression and N-terminal GST tag purification.
CDI uses its Arrayjet Ultra Marathon II non-contact piezoelectric (inkjet) printer to manufacture microarrays with significant performance advantages over contact print methods used in older-generation protein microarrays:
• precise spot morphology (see figure, above)
• Up to 1000 array print batch sizes for large cohort analysis
• rapid production of custom control configured arrays
• reduced inter- and intra-array variability
• greater data reproducibility (see below)
After purification, the GST-tagged proteins are piezoelectrically printed on glass slides in duplicate, along with control proteins (GST, BSA, Histones, IgG, etc.). Slides are barcoded for tracking and archiving. Each microarray batch is routinely evaluated by anti-GST staining to demonstrate quality of expression and printing. Slides can be PATH™ nitrocellulose or SuperEpoxy2™.