Protein arrays LEAPS technology An array of 110 different antibodies incubated with various levels...
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Transcript of Protein arrays LEAPS technology An array of 110 different antibodies incubated with various levels...
Protein arrays
An array of 110 different antibodies incubated with various levels of the fluorescently labelled cognate antigens in a serum background. (Courtesy Dr Brian Haab, The Van Andel Research Institute, Grand Rapids, MI, USA)
Introduction
Detect proteins
Monitor their expression levels
Investigate protein interactions and functions
Production of a Protein Array
Coated surface Robotic proteinapplication
Arrayedproteins
Incubation withfluorescent
labelled sample
Scan
Defining characteristics
Protein arrays are solid-phase ligand binding assay systems using immobilised proteins on surfaces which include glass, membranes, microtiter wells,
mass spectrometer plates, and beads or other particles.
highly parallel (multiplexed) and often miniaturised (microarrays, protein chips).
Rapid
Automatable
Capable of high sensitivity
Economical on reagents
Giving an abundance of data for a single experiment.
Advantages
Areas of application1. Diagnostics: detection of antigens and antibodies in blood samples;
profiling of sera to discover new disease markers; environment and food monitoring.
2. Proteomics: protein expression profiling; organ and disease specific arrays.
3. Isolation of individual members from display libraries for further expression or manipulation: selection of antibodies and protein scaffolds from phage or ribosome display libraries for use in capture arrays.
4. Protein functional analysis: protein-protein interactions; ligand-binding properties of receptors; enzyme activities; antibody cross reactivity and specificity, epitope mapping.
Protein sources
Cell-based expression systems for recombinant proteins
Purification from natural sources
Production in vitro by cell-free translation systems
Synthetic methods for peptides
Many of these methods can be automated for high throughput production. For capture arrays and protein function analysis, it is important that proteins should be correctly folded and functional; this is not always the case, e.g. where recombinant proteins are extracted from bacteria under denaturing conditions. Nevertheless, arrays of denatured proteins are useful in screening antibodies for cross-reactivity, identifying autoantibodies and selecting ligand binding proteins.
Formats and surfaces
Miniaturisation of familiar immunoassay methods such as ELISA and dot blotting.
Commonly used physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads.
Company Architectures
GyrosCD centrifugation devices based on developments
in microfluidics
Biotrove microchannels in a plate
Zyomyx 3D posts on a silicon surface
Bio-Rad colour coding for microbeads
Quantum Dots semiconductor nanocrystals
Smartbeads barcoding for beads
Nanoplex Technologies
multimetal microrods
BioArray Solutions
beads assembled into planar arrays on semiconductor chips
Protein immobilisation considerations
Variables in immobilisation of proteins include: The coupling reagent The nature of the surface being coupled to
The properties of a good protein array support surface are: Should be chemically stable
before and after the coupling procedures, Allow good spot morphology
Display minimal nonspecific bindingNot contribute a background in detection systems Be compatible with different detection systems.
The immobilisation method used should be:
Reproducible
Applicable to proteins of different properties (size, hydrophilic, hydrophobic)
Amenable to high throughput and automation
Compatible with retention of fully functional protein activity
Protein immobilisation considerations
Company DetectionFrom DNA array different fluorophores (e.g. Cy-3, Cy-5)
PerkinElmer Lifesciences
tyramide signal amplification (TSA)
Zeptosens planar waveguide technology
Bio-Rad phycoerythrin as label [Luminex]
Molecular Staging rolling circle DNA amplification
HTS Biosystems, Intrinsic Bioprobes
surface plasmon resonance
Ciphergen, Intrinsic Bioprobes
mass spectrometry
Genicon Sciences
BioForce Laboratories
resonance light scattering
atomic force microscopy
Large-scale protein arrays
Incubation of the slide with anti-RGSHis antibody reveals the recombinant human proteins
Detected with a Cy3-labeled secondary antibody.
Protein array containing 192 purified human proteins on glass.
Challenges and Bottlenecks