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Real-time label-free analysis of pre-immunized (grey arrows) and post-immunized (green arrow) mouse sera exposed to a peptide array. Note the faster association of the immune serum antibodies to peptide 148 compared with the ovalbumin used for conjugation to this immunogen. Inset shows the array image at the end of the experiment

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Image from the application note showing streptavidin binding to biotin probe regions (orange) but not to the PEG controls in between (false color).

Ligands such as biotin can be covalently immobilized on SpotReady™ chips. Such ligand arrays can be used for many purposes, including:

• To monitor the binding of proteins to the ligand(s), or
• To immobilize proteins or other biomolecules that bind to the ligand; or, in the case of biotin surfaces
• To prepare a streptavidin surface on which biotinylated probes may be immobilized.

As an example, this Application Note details how to prepare an array with a biotin surface on some spots and PEG controls on others on the same array. The protocol details how to monitor binding of analyte to the array using the SPRimager®II.

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Image from the application note showing biotinylated oligo binding to streptavidin probe regions (bottom two rows) but not to the PEG controls (second and third rows) nor to streptavidin pre-blocked with biotin (top row).

You can attach streptavidin covalently to SpotReady™ chip surfaces. The attachment strategy uses maleimide-modified streptavidin, and is readily adaptable to creating surfaces from any maleimide-modified protein.

The streptavidin surface generated is convenient for making arrays with biotinylated probes such as biotin-conjugated antibodies, biotinylated oligonucleotides, or biotinylated peptides.

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Array image and corresponding histogram from the application note. Interferon binds to the anti-interferon probe regions (columns 1 & 2) but not to the anti-TNF controls (columns 3 & 4) nor to the streptavidin control (column 5).

It is straightforward to make antibody arrays using commercially available biotinylated antibodies. The antibodies are simply spotted onto streptavidin-coated SPRchip™ or SpotReady™ chip substrates. Specific binding of antigen to the corresponding antibody is then monitored on the SPRimager®II. The application note data confirm that immobilized antibody is active and its recognition specificity is retained.

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Graph from the application note showing the annealing over time of DNA 1 target (top 3 lines) to its complementary probe. The noncomplementary DNA 2 probe regions (bottom 3 lines) of course do not bind the DNA 1 target.

You can make DNA arrays on SpotReady™ chips using thiol-modified DNA oligonucleotides, and monitor specific annealing of complementary DNA target over time. The protocol also explains how end-point measurements may be made using the SPRimager®II.

The method presented for monitoring DNA annealing over time is readily applied to monitoring any molecular interactions. Plus, the method for making arrays with thiolated DNA oligos can be used to make arrays with any thiol modified probe, including thiolated RNA oligomers and peptides synthesized with a terminal cysteine.

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Array image from the application note showing E. coli RNA polymerase binding to immobilized anti-RNA polymerase mAb probes. Each mAb is spotted in triplicate, and the RNA pol antigen binds with varying affinities, yellow indicates highest signal, blue lowest (false color).

You can make arrays of mouse monoclonal antibodies on SpotReady™ chips so that the antibodies are oriented with the antigen binding site exposed. The method first attaches goat anti-mouse antibodies to a streptavidin surface. The monoclonal antibodies are then spotted onto the goat anti-mouse layer. The resulting array maximizes access of the monoclonal antibody recognition regions to the antigen analytes.

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Array image and corresponding histogram quantifying BHK21 cell binding to a protein ligand array. Strongest signals are observed for bFGF probes, with minimal signal for cytochrome C controls.

Protein arrays can be used to analyze not only molecules in solution, but also molecules in their natural state on cell surfaces. This application note introduces a very simple, broadly applicable method for making protein arrays, in this case using proteins that may act as ligands for cell receptors. The SPRimager®II system is then used to monitor how well BHK21 cells attach to the ligand array. Strongest signals are observed for bFGF, since BHK21 cell surfaces express plentiful bFGF receptor.

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Graph from the application note quantifying equilibrium binding of antigen to an antibody array. The surface coverage curves for purified mAb and the corresponding ascites fluid are indistinguishable, resulting in the same affinity value for ascites and pure mAb.

You don’t have to purify monoclonal antibodies to use them as probes on antibody arrays. When you spot purified monoclonal antibody (mAb) and its corresponding ascites fluid on the same array, the affinities for antigen are indistinguishable—i.e. ascites fluid probes yield the same affinity values as purified antibody probes.

The method used here to measure affinities monitors equilibrium binding of antigen over a range of concentrations, and fits the data to a Langmuir isotherm. Collecting measurements at eqilibrium rather than measuring on-rates and off-rates directly helps to make the method robust when using impure components.

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The top array image shows the DNA oligo probe layout, either DNA 1 (blue) or DNA 2 (gold). The middle image shows the change in reflectivity of the array after exposure to the complement to DNA 1. Only DNA 1 probe spots show increased reflectivity, as only probe 1 binds DNA1 to become double stranded. DNA 2 probes remain single stranded. The bottom array image shows the change in reflectivity after further exposure to Single-Strand Binding protein (SSB). SSB binds only to ssDNA, and not to dsDNA, so it binds only to DNA 2 probes, and not to the DNA 1 probes, which are now dsDNA.

This example illustrates how you can monitor multiple interactions involving molecules of different chemical composition. An array was made using replicates of just two noncomplementary DNA probes, 24-nt long. When the complement to DNA 1 was exposed to the array, it hybridized specifically to DNA 1 as expected, resulting in an array with dsDNA probes for DNA 1 and ssDNA probes for DNA 2. Then, when SSB protein was exposed to the array, SSB bound only to DNA probe 2, because only this probe is single-stranded. You can view how this experiment was done by watching this 5MB movie.

There are countless other ways you could use the SPRimager®II to monitor multiple binding events.

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Real-time monitoring of target DNA-dependent loss of SPR signal on AmpliFast™ RNA oligo arrays. The graph shows the signals for three oligo probes on the array: Kan RNA (triangles), Luc RNA (squares) and Luc DNA (circles). After exposure of the array to DNA complementary to Kan RNA, only the Kan RNA probe is degraded.

GWC’s AmpliFast™ technology provides a completely label-free method of nucleic acid detection that requires no amplification of target DNA. Instead, amplification occurs on the array, letting you monitor the reaction in real time.

AmpliFast™ arrays are made using RNA instead of DNA oligos. When complementary target DNA hybridizes to the array, it forms an RNA-DNA duplex. The RNA strand in this duplex is specifically sensitive to degradation by RNase H. When monitored on the SPRimager®II, this degradation leads to a loss of SPR signal at a rate proportional to the concentration of DNA target. Isothermal, linear amplification occurs automatically, as the target DNA is not degraded, and can therefore repeat the cycle of annealing to RNA probes, leading to further degradation of probe molecules.

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