Protein immobilization images

The 3-D NPH technology was applied to immobilize streptavidin (SA) onto Au-coated surface for surface plasmon resonance imaging (SPRi). By using 3-D NPH method, it was possible to improve the sensitivity of protein-protein interactions drastically comparing to the conventional protein immobilization method. 

Fig. 3.6 Schematic diagram of a multi-spot plate assay for four human cytokines. Each spot within each well of the multi-well plate contains capture antibody specific for one cytokine. (Inset) Images of the ECL emitted from assays and protein immobilized on carbon electrode surface (Reprinted with permission from Ref. [10]. Copyright 2008 American Chemical Society)...

The shift of the amide I mode (FTIR spectra) from 1657 to 1646 cm-1 was attributed to a change in the a-helix native structure to fl-sheets, secondary structure conformations. Atomic Force Microscopy (AFM) images display the coating of the manganese oxide surface as well as the unfolding in a ellipsoidal chain of the protein molecules after adsorption and immobilization on the surface. 

Figure 4 Quartz microbalance electrode with a protein A-HRP conjugate immobilized on the gold surface, (a) Transmitted light image (b) chemiluminescent signal after addition of CL substrate (c) 3-D display of the light signal spatial distribution.

Duplicating the GFP-H2B experiments with both GFP-H3 and GFP-H4 vectors, very little H3 and H4 exchange outside of S phase was observed. Fluorescence of GFP-H3 and GFP H4 in HeLa cells in G1 recovered rapidly. The extremely rapid recovery rate is similar to that of a diffuse soluble protein, indicating that at this stage of the cell cycle, GFP-tagged H3 and -H4 are not incorporated into chromatin. FRAP experiments of transfected cells in S or G2 show that there is very little recovery of GFP-H3 or -H4 fluorescence. The fluorescence imaging indicates that once the H3 and H4 proteins are incorporated into the chromatin, they are essentially immobile for the remainder of the cell cycle. Unlike histones H2A and H2B, which associate as dimers in the nucleosome histone octamer, there is very little exchange of the components of the H3/H4 tetramer. 

Most recently, we reported small molecule arrays on photoaffinity crosslinker coated gold surfaces (17). The small molecule arrays were fabricated by photoreaction, and then analyzed by SPR imaging technique. The small molecules don t have to be modified chemically for immobilization. The small molecules, which can interact with a target protein, can be screened by this methodology. Therefore, the integration of photoaffinity small molecule array and SPR imaging technique can be the first step of reverse chemical genetics. 

Figure 11.2 The image in the upper left panel shows a snapshot of several individual protein molecules immobilized in a gel. Each protein undergoes conformational fluctuations that can be monitored by a fluorescent probe. The fluorescent signal from a single protein molecule, as a function of time, is recorded in the time trace shown in the lower left panel. On the right, the experimental situation and the fluorescent time trace are idealized as a two-state conformational transition process as given in Equation (11.5), with A representing the darker state and B representing the brighter state. Image and data in left panel obtained from Lu et al. [133], Reprinted with permission from AAAS.

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