Protein-detected techniques

Huang, Y. Y, H. Y. Hsu, and C. J. Huang. 2007. A protein detection technique by using surface plasmon resonance (SPR) with rolling circle amplification (RCA) and nanogold-modified tags. Biosens. Bioelectron. 22 980-5. 

A new approach to study root exudation of distinct compounds in soil-grown plants uses inoculation of roots with genetically engineered reporter bacteria, which are able to indicate the presence of particular compounds by indicator reactions, such as production of ice-nucleation proteins. This technique has been employed to detect the release of amino acids from roots of soil-grown A vena harbata (56). 

BRET [31, 32]), lock-in detection techniques exploiting optical switches [33], and schemes for alternating D/A excitation (ALEX [34]). The increased attention to quantitative FRET imaging encompasses the use of polarization [35-39], the perennial issue of calibration and standards [40-44], and practical guides to operational principles and protocols ([45, 46] and other references above). The fundamental distinctions between the requirements for live and fixed cell imaging cannot be overemphasized, as is exemplified in a report of erroneous FRET determinations with visible fluorescent proteins (VFPs) in fixed cells [47],... 

Silver also binds to proteins, an observation that forms the basis of an extremely sensitive method of protein detection. This technique is used extensively to detect proteins in electrophoretic gels, as discussed in the next section. 

A modified version of 2DE and gel image analysis, with silver staining, autoradiography, and protein identification and measurement of peptide mass, uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as a rapid and sensitive technique for identifying peptides. MALDI-TOF-MS applies well to protein detection in biological fluids.56 A second advantage of this technique is... 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

The search for more rapid and sensitive methods of protein detection after electrophoresis led to the development of fluorescent staining techniques. Two commonly used fluorescent reagents are fluorescamine and anilinonaphthalene sulfonate. New dyes based on silver salts (silver diamine or silver-tungstosilicic acid complex) have been developed for protein staining. They are 10 to 100 times more sensitive than Coomassie Blue (Fig. 4.7). 

Very few immunosensors are commercially available. The commercial immunosensors are either the detector or bioanalyzer types. The PZ 106 immunosensor from Universal Sensors Inc. (New Orleans, LA) has been used as a detector to measure antibody-antigen reaction. Ohmicron (Newtown, PA) developed a series of pesticide immuno-bioanalyzers that have been used in field tests. Pharmacia Biosensor USA (Piscataway, NJ) recently introduced BIAcore immunodetection system. A combination of a unique flow injection device and surface plasmon resonance (SPR) detection technique provides a real time analysis. A carboxylmethyldextran layer added to plasmon generating gold film is a hydrophobic, activatable, and flexible polymer that provides high antibody and low non-specific bindings. System demonstration at the Institute of Food Technologists (IFT) 1994 meeting in Atlanta drew attention of food scientists. It should easily be adapted for food protein characterization. 

---