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Protocol for Preparation of Streptavidin-coated Slides based on SCHOTT Nexterion 3-D thin-film Microarray Slides

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Protocol for preparation of streptavidin-coated slides at point of use by Prof Jonathan Blackburn; University of Cape Town

Protocol for preparation of streptavidin-coated slides at point of use by Prof Jonathan Blackburn; University of Cape Town

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  • 1. Protocol for preparation of streptavidin-coated slides at point of use<br />Prof Jonathan Blackburn; University of Cape Town<br />November 2010<br />Materials<br />1. Steptavidin (Sigma or other equivalent supplier)<br />2. Nexterion Slide P or H (SCHOTT http://www.schott.com/nexterion/)<br />3. Lifterslips (Nunc, USA)<br />4. Ethanolamine (Sigma)<br />5. Wash buffer: 20 mM KH2PO4 , 0.2 mM EDTA, 5% glycerol, pH 7.4<br />6. Cy3-labelled, biotinylated BSA (made in-house)<br />7. PBST: 1.5 mM KH2PO4, 4.3 mM Na2HPO4, 137 mM NaCl, 3 mM KCl, 0.1% (v/v) Tween20, adjust to pH 7.4 with HCl.<br />Preparation of streptavidin-coated slides for microarray printing<br />Equilibrate a Nexterion Slide P (or H) microarray slide to room temperature and remove from the foil package.<br />Make up a 1mg/ml streptavidin solution in 150mM Na2HPO4 buffer (pH 8.5).<br />Place a glass microarray ‘lifterslip’ over the microarray surface and pipette 60µl of the streptavidin solution along the edge of the lifterslip such that the solution is drawn under the cover slip uniformly by capillary action.<br />Leave for 1hr at room temperature in a humidified chamber.<br />Remove the lifterslip and wash the slide for 1hr at RT in 10ml 150mM Na2HPO4 buffer (pH 8.5) containing 50mM ethanolamine to deactivate any remaining amine-reactive groups. <br />Wash the slide for 3 x 5 min in 10ml wash buffer and then for 5 min in 10ml water. <br />Place the slide in a 50 ml Falcon tube and centrifuge at 250 rcf for 1 min at 20°C to spin dry.<br />Place steptavidin-coated slides into slide boxes, seal in Ziploc bags and store at -20°C.<br />Determining the uniformity of the streptavidin coating<br />
    • Equilibrate one streptavidin-coated slide to room temperature and wash for 1 min in 150mM Na2HPO4 buffer (pH 8.5) then centrifuge briefly (250 rcf; 1 min) to remove excess surface liquid.
    Make up a 1g/ml solution of Cy3-labelled, biotinylated BSA in 150mM Na2HPO4 buffer (pH 8.5).<br />Place a glass microarray ‘lifterslip’ over the microarray surface and pipette 60µl of the Cy5-labelled, biotinylated BSA solution solution along the edge of the lifterslip such that the solution is drawn under the cover slip uniformly by capillary action.<br />
    • Leave for 1hr at room temperature in a humidified chamber.
    • 2. Remove the lifterslip and wash the slide for 3 x 5 min in 10ml PBST, then centrifuge briefly (250 rcf; 1 min) to remove excess surface liquid.
    • 3. Scan the slide in a fluorescence microarray scanner (excitation 550nm; emission 570nm) and determine the uniformity of fluorescence across the slide.
    Notes<br />We and others have found that organic polymer coatings, such as those based on dextran or polyethylene glycol (PEG) (either in the form of long chain PEGs or of short chain PEGs supported on a self-assembled monolayer), are considerably superior to proteinaceous blocking agents such as bovine serum albumin or powdered milk in reducing the non-specific binding background in surface-based assays (1); a number of commercially-available surfaces conform to this specification.<br />One such commercially available PEG-based streptavidin-coated surface that works well in resisting non-specific protein binding is the Nexterion Slide H-S from SCHOTT, although we have also used other streptavidin-coated glass or plastic surfaces with similar ‘non-stick’ properties. However, most if not all pre-coated streptavidin slides must be shipped on dry-ice in order to preserve the integrity of the streptavidin layer and the shipping costs can therefore be substantial, depending on location. In order to circumvent this cost issue, we have found that we can custom coat amine-reactive slides (for example the PEG-based, NHS-activated Nexterion Slide P from SCHOTT, which ships at room temperature and therefore at significantly lower cost) with streptavidin at point of use. Importantly, we have found that our home-made slides show as good uniformity of the streptavidin coating as commercial, pre-made streptavidin surfaces (data not shown); furthermore, we found that utilising an automated hybridisation station in an effort to gain greater control over the streptavidin coating process gave no obvious advantage over the manual process described here (data not shown). <br />References<br />1. Zheng, J. Li, L. et al. (2005) Strong Repulsive Forces between Protein and Oligo (ethylene glycol) Self- Assembled Monolayers: A Molecular Simulation Study. Biophys. J. 89, 158-166.<br />