Protein sensitivity limitation

The disadvantages associated with HRP are several. The enzyme only contains two available primary e-amine groups—extraordinarily low for most proteins—thus limiting its ability to be activated with amine-reactive heterobifunctionals. HRP is sensitive to the presence of many antibacterial agents, especially azide. It also is reversibly inhibited by cyanide and sulfide (Theorell, 1951). Finally, while the enzymatic activity of HRP is extremely high, its useful lifespan or practical substrate development time is somewhat limited. After about an hour of substrate turnover, in some situations its activity can be decreased severely. 

The most frequently used protein assay is based on a method after Bradford (Bradford, 1976), which combines a fast and easily performed procedure with reliable results. However, the Bradford assay has sensitivity limitations and its accuracy depends on comparison of the protein to be analyzed with a standard curve using a protein of known concentration, commonly bovine serum albumin (BSA). Many commercially available protein assays such as those from Pierce or BioRad rely on the Bradford method. The assay is based on the immediate absorbance shift from 465 nm (brownish-green) to 595 nm (blue) that occurs when the dye Coomassie Brilliant Blue G-250 binds to proteins in an acidic solution. Coomassie dye-based assays are known for their non-linear response over a wide range of protein concentrations, requiring comparison with a standard. The dye is assumed to bind to protein via an electrostatic attraction of the dye s sulfonic groups, principally to arginine, histidine, and lysine residues. It also binds weakly to the aromatic amino acids, tyrosine, tryptophan, and phenylalanine via van der Waals forces and hydrophobic interactions. 

The high sample demands and low-throughput of LC-MS methods have led to the creation of a capillary electrophoresis (CE) platform for ABPP [48]. Proteomes are labeled with a fluorescent probe, digested with trypsin, and enriched with antifluorophore antibody resins. Use of CE coupled with laser-induced fluorescence (LIF) detection to analyze the enriched peptides resulted in far superior resolution to ID SDS-PAGE, particularly for enzymes that share similar molecular masses. Sensitivity limits of 0.05-0.1 pmol/mg proteome, negligible sample requirements (—0.01—0.1 pg proteome), and the ability to perform rapid CE runs in parallel with 96-channel instruments, make CE-based ABPP a potentially powerful technique. One drawback is that the identities of the probe-labeled proteins are not immediately apparent, and correlated LC-MS experiments must be performed to assign protein identities to the peaks on the CE readout. 

Cadmium is characterized by high sensitivity (limit 0.03 ppm) (G2) and by complete atomization in the air-coal gas flame. Willis (W14) showed that cadmium when added to urine could be determined by aspirating the mine directly normal urine levels were at the sensitivity limit of the method, but applications of this technique are certainly feasible in toxicological work. The recent demonstrations of a specific cadmium-containing protein (K2, K3) adds importance to the availability of an atomic absorption method for cadmium. 

Radioactively labelled proteins may be visualized without staining by autoradiographic methods which were first introduced by Becquerel and Curie in their discovery of the phenomenon of radioactivity (13.), or f 1 uorographic techniques for some of the weak beta emitters, such as tritium(M ). If the proteins are radioactively labled to a high specific activity, they can be detected with sensitivities equal and often better than those obtained by the most stains. However, the use of radioactively labled proteins is limited as it is difficult to achieve high specific activities in animal studies and unethical in reseach involving humans. 

Although flat or two-dimensional (2D) surfaces are used for various apph-cations within the DNA and protein microarray area, practical uses for SPR detection were originally limited. This can be attributed to the sensitivity limitations of the technology, despite the relative ease in handhng and the wide variety of developed chemistries based on flat surface structures. Thus, an immobilized monolayer may not give sufficient binding responses under certain conditions, especially if immobilization leads to compromised activity of the immobihzed partner. As described previously, non-specific binding also needs careful consideration. 

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