HuProt™ Human Proteome Microarrays v3.1*

 

This array offers the largest number of human proteins currently available on a single microarray chip, the majority in full length. HuProt™ arrays contain greater than 20,000 unique, individually printed proteins, representing more than 16,152 human genes and covering 81% of the human proteome. The recombinant proteins are expressed in yeast (S. cerevisiae), purified and printed onto coated glass slides in duplicate, along with control proteins.

table1 HuProt

Table 1: Protein class coverage analysis has been performed per annotation in The Human Protein Atlas. As members of these classes overlap the table is not additive.

Specifications:

 

Protein content:

  • 20,000 human proteins, representing > 16,152 human protein coding genes
  • In-depth coverage of major protein classes
  • Expressed in cerevisiae with N-terminal GST-RGS-His6 tag
  • GST-based purification
  • Fluorescent control spots for grid alignment
  • Spotted assay controls: GST dilution series, biotinylated BSA, mouse IgG anti-biotin, rabbit IgG anti-biotin, mouse IgM, BSA, buffer

Array layout:

  • Each protein spotted in adjacent duplicates, vertically staggered
  • Printed area: 5 mm x 52.5 mm, 4 x 6 subarrays
  • Corresponding .gal file with spot locations and identities provided with each array batch
  • Customized inclusion of additional proteins on request

Slides:

  • SuperEpoxy2™ (Non-contact printer) slides: surface polished to ± 20 Å
  • Slide dimensions 25 mm x 76 mm x 1 mm
  • Barcoded for unambiguous and automated tracking

 

Applications:

 

Autoantibody screening in serum samples:

Autoantibodies, leading to immune reactivity against specific tissues, are highly associated with the pathology of autoimmune diseases. Identifying which antigens are the target of autoantibodies can be the first step to understanding the underlying disease mechanisms, as well as to disease stratification. Autoantibodies have also gained prominence as early diagnostic biomarkers in cancer, where they may emerge as an effect, rather than as the cause of the disease. HuProt™ arrays provide the option to discover and identify signature autoantibodies through interaction with their arrayed antigens.

HuProt cut out

Figure 1: Example of human serum probed on HuProt microarray revealing differential affinity for human proteins.

Comprehensive antibody reactivity profiling

Cross-reactivity is an often-overlooked issue with antibodies, typical QC involving proof of binding to the intended target, but neglecting broad screens for potential cross-reactivity.  Screening your antibody on HuProt™ v3.1 arrays allows to identify potential cross-reactivity to 81% of the human proteome. This is of particularly high value for the characterization of therapeutic or diagnostic candidate antibodies, where cross-reactivity can provide an early indication of possible off-target effects in vivo, or of potentially misleading diagnostic readouts. By eliminating candidate antibodies which show cross-reactivity from the development process, significant savings in cost and time can be achieved. Analogous to classical antibodies, alternative binder types can also be assayed on HuProt arrays. HuProt human proteome microarray is a powerful tool for simultaneously assaying antibody specificity against the largest collection of human proteins in the world. The International Working Group for Antibody Validation has recommended guidelines for use of protein microarray as a method for confirmation of target binding. (1,2)

barplots antibodies

Figure 2: Bar plot graphic of top 20 hits on HuProt Microarray. Comparison of two commercial monoclonal Antibodies with the same target protein from different provider. HuProt array analysis difference in binding affinity between the two antibodies. Antibody on the left shows unspecific binding and a low binding affinity for intended target. Antibody on the right side shows high specific affinity for target and almost no unspecific binding.

Protein-protein interactions

Interactions between proteins are at the center of biochemical pathways. HuProt™ arrays provide a platform for screening proteins for binary interactions with the world’s largest collection of human proteins immobilized on a single microarray.  Over 20,000 potential interactions are assessed in a single experiment. Compared to pulldown methods detecting endogenous interaction partners, the potential interaction partners are present on the array in less diverse protein concentrations. This facilitates detection of those interactors with low abundance in the cellular system.

Our Service with HuProt™:

 

  • Individual consultation to determine optimal assay design and optimization
  • Sample handling and array processing
  • Array scanning and data acquisition
  • Data analysis
  • HTML based report, catered to the requirements of customer’s project

References

(1) Ramos, Pedro, Lehy, Andrew, Pino, Ignacio, and Paschke, Scott, White Paper CDI Laboratories April , 2016, “Antibody Cross-Reactivity Testing Using the HuPro Human Proteome Microarray”

(2) Uhlen, Mathias et al. Nature Methods Vol 13. No. 10, October 2016. “A proposal for validation of antibodies”

*manufactured by CDI Laboratories Inc.