HuProt™ Human Proteome Microarray
With more than 21,000 GST-purified proteins, there has never been a protein library as comprehensive or thoroughly validated as HuProt™.REQUEST INFO
Most fluids contain antibodies and we can analyze them (i.e., peripheral serum/plasma, cerebrospinal fluid, tissue lysates, stool, saliva), or in a synthesized sample, for any isotype (i.e., IgM, IgG, IgA, IgD, IgE). Secondary antibody binding is then quantified using fluorescence intensity measurements of features on the array. We’ll provide you with those RAW readouts with normalized readouts and group-comparison statistics (optional). HuProt arrays also allow for pan-isotype autoantibody biomarker profiling versus properly folded, three-dimensional human proteins.
There’s more to HuProt than serum profiling. We’ve profiled nucleotide binding, antibody specificity, small molecule binding, protein-protein interactions, and more. If you can dream up a study, we can help you get it done. HuProt contains >21,000 unique proteins and was built during the NIH Protein Capture Reagents program.
HuProt is an incredibly sensitive antigen matrix platform that provides quantitative binding interaction data from a few microliters of sample. It has been used to identify everything from the immunologic fingerprint of cancer patients destined for checkpoint blockade toxicity to novel protein-protein interactions.
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.
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.
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.
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.