Protein multiplex profiling as simple as western blot

In one of my previous posts, I wrote about high density glass slide antibody arrays services.

Here, I’d like to focus on another antibody microarray product line, the C-series from Raybiotech, based on a membrane antibody array technology. Similar to western blotting – therefore very easy to process – they are suitable to most research laboratories for targeted protein profiling.

The C-series allow a broader view of protein expression than can be obtained with single-target Western blots. Moreover, antibody array screening improves the chances for discovering key factors, disease mechanisms or biomarkers related to cytokine signaling.

The C-Series Arrays utilize the sandwich immunoassay principle, wherein a panel of capture antibodies is printed on a nitrocellulose membrane solid support (usually 2.5 cm x 3 cm). The array membranes are processed similarly to a Western blot (chemiluminescent readout). Signals are then visualized on x-ray film or a digital image, allowing densitometry data collection and calculation of fold-changes for each detected protein. The entire procedure can be completed in 1 day, and is simple enough to permit even the novice researcher to successfully collect data with very few pitfalls and little or no optimization.

What are the main features of these membrane arrays?

• Simultaneous detection of multiple proteins
• High specificity (sandwich antibody pair used)
• High sensitivity (pg/ml levels)
• High throughput
• No dedicated equipment required; compatible with any chemiluminescent imaging system
• Easy to use, no training required
• Results can be obtained in 1 day

For which research applications?

• High-throughput profiling of cytokine expression
• Identifying potential molecular targets for drug development
• Identifying the molecular mechanisms of drug action
• Identifying crucial factors involved in disease processes
• Discovering biomarkers for disease management
• Discovering expression patterns for molecular classification of diseases

A featured reference from Nature Immunology:

Mouse Cytokine Array C4 in Nature Immunology
Kashiwagi M., Hosoi J., Lai J., et al. Direct control of regulatory T cells by keratinocytes.
Nat Immunol. 2017 Mar;18(3):334-343. doi: 10.1038/ni.3661

Understanding how local environmental changes affect the immune response is a principal element in immunology. Specifically, how does this environment impact regulatory cells, what cells take part in the process, and what secreted factors are important remain an intense focus. Here, this group focused in on how keratinocytes secrete factors that control the activation state of regulatory T cells. In order to determine what factors were secreted, they began by using RayBiotech’s Mouse C-Series array 4 to probe for a broad array of potentially secreted cytokines. Thymic stromal lymphopoietin (TSLP) was found to regulate activity in Treg’s, whereby the inability to sense TSLP in the system would result in an uncontrolled pro-inflammatory response. Keratinocytes, and TSLP, therefore play a key role in inflammatory situations, support regulatory T cell suppressive responses, and are critical to restoration of homeostasis in the system.

If you feel this kind of studies and the microarray tool to go with it can be of interest to you, do not hesitate to contact me to get more information on the technology or targets available, or if you want to outsource your project by experts. We run the glass slide equivalent to the C-series (fluorescence detection) in our own laboratories in Le Perray, for either profiling or multiplex quantitation projects.

Isabelle Nobiron
Written by Isabelle Nobiron
Isabelle is a Product Manager at tebu-bio, and also the company's ISO Quality Manager.