Glass slides modification with

Figure 17.3 Maleimide-modified glass slides (1) can be derivatized using two chemoselective ligation reactions to create biotin modifications. In the first step, alkyne-PEG4-cyclopentadiene linkers (2) are added to the maleimide groups using a Diels-Alder reaction. In the second reaction, an azido-PEG4-biotin compound (3) is reacted with the terminal alkyne on the slide using click chemistry to result in another cycloaddition product, a triazole ring.

Fluoro-Jade staining was performed as described in Schmued and Hopkins (2000) with modifications (Butler et al. 2002). The sections were wet mounted onto microscope glass slides and air-dried for 30 min at 37°C in an oven. Then, the sections were pretreated for 5 min in absolute alcohol, followed by 3 min in 70% ethanol, 3 min in 50% ethanol, and 5 min in distilled water. The slides were then immersed in a solution of 0.05% KMn04 for 30 min at room temperature, and stained for 30 min in a solution of 0.001% Fluoro-Jade B (Chemicon, Temecula, CA, USA) in 0.1% acetic acid. Finally, the slides were then rinsed in distilled water, dried, cleared in xylene, and coverslipped. 

The functional microarray typically consists of a collection of full-length functional proteins or protein domains printed on glass slides that are then exposed to a protein preparation from a cell that represents the entire proteome of that cell. This method is useful in determining protein-protein interactions. In addition, this method is useful in predicting the interaction of proteins with DNA, RNA, phospholipids, and small molecules. RPA includes glass slides on which a cellular protein preparation is fixed and then probed with a known antibody. This method helps in identifying the proteins that are altered and cannot bind with a known antibody in the proteome of diseased cell types. This method also identifies the proteins that are altered as a result of phosphorylation or other posttranslational modifications in normal and disease conditions or under growth conditions. 

Enzymes are robust biological systems. They can be stored in the refrigerator or in the freezer for extended periods of time. Their activity under homogenous or immobilized conditions can be measured spectroscopically and electrochemically (140-142). There are no special equipment needs for SECM studies as the solid support modification is often done on electrodes or glass slides that can easily be combined with a conventional SECM setup. Most of the equipment needs are related to the patterning or immobilization of the enzymes onto solid supports. 

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