Detection of target proteins

Notwithstanding the aforementioned difficulty in detecting specific target proteins other than the types normally observed in the taxonomic fingerprints from whole bacteria MALDI spectra (i.e., ribosomal proteins), some other target proteins and protein-like materials have been studied directly from whole cells. For example, Lantibiotics, antimicrobial peptides secreted by Gram-positive bacteria have been detected directly from whole bacteria by MALDI-TOF MS.51 The lantibiotics nisin and lacticin 481 were detected from whole cells and crude supernatants. Surprisingly, better results were reported from whole cells than the supernatants. In this study the presence of variants 

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Scheme 13 Illustration of the specific detection of target proteins by using the complex of a cationic polythiophene(2I)/dye-labeled ssDNA aptamer on glass slides. Reproduced with permission from [102]...

Fig. 7 A schematic illustration of the enzymatically amplified SPRi detection of target proteins (from [44])...

Figure 14 6 Silver-stained SDS-PAGE gel of PatA binding proteins. Lane 1, sample 1 nonspecific proteins captured by the streptavidin-agarose resin Lane 2, sample 2 proteins affinity captured by the presence of B-Pat A Lane 3, sample 3 affinity capture of target proteins was blocked by prior addition of free PatA before incubation with B-PatA. The two arrows point to two proteins specifically detected in sample 2 versus sample 1, which were also lost due to competition in sample 3, with apparent molecular weights of 38 and 48 kDa.

Furthermore, since the cell growth arrest is often linked to cell death. The annexin V staining positive cell or the amount of DNA fragmentation assessed by TUNEL and FACS analysis has been interpreted as indicative of apoptosis. The HDACI-induced apoptosis can also be determined by Western blotting of target proteins, detection of mitochondrial membrane potentials, activation of caspases and their substrate cleavages in a dose- and time-dependent manner. 

Besides normal ECL detection reagent, ECLprime (Amersham) or other similar products from other vendors (e.g., Millipore) are now available new-generation ECL products for chemiluminescent detection which provide higher sensitivity over the normal ECL reagent. If the concentration of target protein is too low and the primary antibody is limited, these products may be considered as an alternative. 

Label-free detection of ligand-aptamer interaction was also demonstrated by means of impedance spectroscopy technique [52,53]. Simultaneously, Radi et al. [52] and Rodriguez et al. [53] reported application of Faradaic impedance spectroscopy (FIS) in detection of interaction of proteins with DNA aptamers. The detection method is based on the measurement of resistance in presence of redox mediator Fe(CN)6-In absence of target protein, the negatively charged aptamer repulse the redox mediator molecules from the sensor surface. In a paper by... 

Serebriiskii, I.G., Mitina, O., Pugacheva, E.N., Benevolenskaya, E., Kotova, E., Toby, G.G., Khazak, V., Kaelin, W.G., Chemoff, J. and Golemis, E.A. (2002) Detection of peptides, proteins, and drugs that selectively interact with protein targets. Genome Res. 12, 1785-1791. 

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