The Kinex™ Antibody Microarray (KAM) Services for signal transduction protein profiling are rapid, convenient and extremely cost-effective solutions for the broad discovery of productive research leads such as biomarkers and extensive characterization of specific proteins, identification of kinase and phosphatase substrates, and can be adapted for detection of kinases sensitive to inhibitors.

Our Kinex™ antibody microarray track the differential binding of dye- or biotinlabelled proteins in lysates prepared from cells and tissues. The results can yield useful insights into differences in protein expression, covalent modifications such as phosphorylation, and protein-protein interactions, and define antibody reagents that can be used to follow up on these findings with other immunological techniques such as Western blotting and immunohistochemistry.

Kinex™ KAM antibody microarrays permit the simultaneous probing of hundreds of target proteins and phosphorylation sites with as little as 25 μg of crude cell or tissue lysate protein. No other proteomics technology, including mass spectrometry, can compete with antibody microarrays for the directed study of target proteins in lysate samples in terms of sensitivity, speed, reproducibility, dynamic range, and cost. For example, an analysis of 5,000 functionally important phosphosites in the PhosphoSitePlus website revealed that approximately half of them were undetected or reported only once or twice across 4,000 high throughput mass spectrometry studies. Therefore, the antibody microarray is a particularly attractive initial route for taking a systems biology, proteomics approach to studying human diseases and experimental model systems.

Presently, Kinexus offers the KAM-2025 antibody microarray in its services and as a stand-alone product in kits. The KAM-2025 chip features 1,047 phosphosite-specific antibodies and 980 pan-specific antibodies for many of the same phosphoprotein targets. About 949 unique human proteins are tracked, of which 380 are catalytic and regulatory subunits of protein kinases and 65 are subunits of protein phosphatases. Each antibody is printed twice per field, which allows for duplicate measurements. About 483 (46%) of the phosphosite-specific antibodies target regulatory phosphorylation sites on protein kinases, and 60 (5.7%) of the antibodies target protein phosphatases. In terms of unique target proteins, these include 282 protein-serine/threonine kinases (including 16 dual-specificity kinases), 82 protein-tyrosine kinases, 114 transcription factors; 63 protein and lipid phosphatases, 66 other metabolic enzymes, 56 adaptor/scaffold proteins, 42 stress and apoptosis proteins, and over 240 other target proteins (exact numbers 14 range between microarray printings). Many of these targets were selected to be especially useful for oncology and neurobiology applications. Presently, we offer services with this microarray that permit detections of changes in protein expression levels and specific phosphosites (KAM-2025) and protein-tyrosine phosphorylation (KAM-2025-pY). Clients should contact us regarding alternative detection formats for other types of covalent modification (e.g., ubiquitination) and protein- and drug-interactions.

With our Kinex™ antibody microarray services, all cell/tissue lysate samples are directly labeled with the same dye mixture or biotinylated and analyzed separately on Kinex™ microarray chips from the same print run. This avoids the misleading results that can arise from the differential binding of different dyes to proteins that is problematic with the two dye competitive system used with other commercial antibody microarrays and the DIGE 2D gel electrophoresis technique.

Non-denatured proteins are typically analyzed on commercial antibody microarrays offered by other vendors. This option is also available with the Kinex™ KAM antibody microarrays. However, there is increased opportunity for false positives and false negatives due to antibody cross-reactivity, antibody competition for same antigen but different epitopes, protein-protein interactions, and blocked epitopes in protein complexes. In addition, the intensity of recorded signals may reflect the size of the target proteins with larger proteins giving stronger signals. From our internal studies with cells from different species, only about a third of the protein changes detected on Kinex™ KAM antibody microarrays are reproduced by immunoblotting.

To reduce the rates of false positives and negatives from the problem of protein-protein interactions, we have added the option of having the lysate proteins subjected to chemical cleavage in a manner that preserves the integrity of most protein phosphorylation sites, especially when this is performed at the time of chemical cleavage. The chemical cleavage effectively eliminates the enzyme activities of kinases, phosphatases and proteases. It should be noted that even with chemical cleavage, some phospho-epitopes may not be accessible due to internal interactions with flanking arginine and lysine residues that can mask the phosphosite from phosphosite antibodies. This issue is evident, for example with the “TEY” dual phosphosites in the ERK1 and ERK2 MAP kinases. Since lysate samples are highly stable at ambient temperatures after the chemical cleavage step, and it is feasible to courier lysate samples to Kinexus for KAM analyses without the need for refrigeration or freezing during shipping.