Over 21,000+ Full-length, Correctly-folded 3D Proteins

Technical Details

With over 21,000 human proteins and protein isoforms, CDI Labs’ HuProt Human Proteome Microarray is the most comprehensive human full-length protein array ever created. This covers over 81% of human proteins in each major functional category of the proteome, as defined by the Human Protein Atlas, and allows hundreds of interactions to be profiled at once in a wide range of applications, including mapping antigen-specific immunity (multi-isotype serum profiling), determining monoclonal antibody specificity, and to study protein-protein interaction, substrate identification, protein-DNA binding, protein-RNA binding, and binding of some small molecules.

The HuProt expression library was created by inserting full-length human open reading frames (ORFs) inserted into a yeast high-copy expression vector that produces GST-His6 fusion proteins when induced in yeast. Each human ORF expression vector was verified for integrity by Sanger sequencing. These sequence-verified expression vectors encoding human ORFs were transformed into yeast to create our master library; this eukaryotic expression system ensures HuProt™ proteins maintain both function and proper conformation.

Full-length synthesized proteins are purified via their GST-His6 fusion tags. These proteins are then frozen in aliquots in a 384-well format for later use in printing on HuProt™ microarrays.

To print individual batches of HuProt arrays, the purified human proteins are then printed as duplicate spot pairs using an Arrayjet UltraMarathon printer (Arrayjet, UK) on PATH nitrocellulose slides (GraceBio, USA). Controls on HuProt include titrated GST protein, titrated human IgG, other human antibody isotypes, histones, mouse and rabbit anti-biotin, mouse IgM, and biotin-tagged control for streptavidin detection. HuProt also contains landmark control spots, including Alex Fluor 555/647 (Jeong et. al., 2012). Success of each batch of HuProt microarrays is validated by anti-GST staining. This confirms full-length GST-His6 fusion proteins were successfully expressed, synthesized, purified, and printed across our clone collection.

Service Details and Data Deliverables

HuProt service involves case and control serum samples diluted 1:1000 and reacted with individual microarrays with BSA blocking. After incubation, samples are stained for one or two desired fluorescent secondary detection antibodies (i.e., IgG / IgA / IgM). Arrays are scanned and raw TIF images are created using Molecular Devices Genepix microarray scanners. Spots are then aligned to CDI library (.gal) files, and spot readings are output as raw Genepix results (.gpr) files. These raw results are then delivered both as .gpr files and combined data tables. If the samples are blinded to CDI Labs, this is the final output. If sample blinding is not a requirement, CDI Labs can additionally prepare a comprehensive sample report with statistical and QC outputs.

Sample Requirements

Human serum or plasmaOne 20 μL per sample

Antibody isolates10 μg per sample (concentration 0.1 mg/mL)

Human (CSF) cerebrospinal fluidMinimum 1.5 mL per sample

Protein / peptide / small molecule10 μg per sample (concentration 0.1 mg/mL)

RNA1 mL per sample (concentration 2 μM)

Other fluids> Please contact us

Cohort balancing (studies >60 samples)

Individual samples are run on individual HuProt slides; these are produced in batches of 600 and our lab team typically processes x60 samples per day. While data from multiple days and print batches is easily pooled by our informatics pipeline, customers running larger studies may wish to consider balancing case/control and replicate samples within and across both processing days and print batches.

Sample Shipping and Sample Return

We will include shipping details in your quote – you must cover the cost of shipping samples to CDI Labs. Typically, after you receive your report, CDI Labs keeps the remaining samples for two months and then disposes of them. When shipping to us, please let us know if you want the remaining samples returned after the study is complete. Return shipping will be charged.