Protein precipitation advantages

Methanol and acetonitrile are the most frequently used water-miscible organic solvents for protein denaturation. The main advantage of using methanol for protein precipitation is that a clear supernatant is obtained and a flocculent precipitate is formed. Acetonitrile, on tire other hand, gives a hazy supernatant with a fine precipitate. The compatibility of tliese solvents with the reversed-phase liquid chromatographic eluents commonly used for separation purposes is an added advantage. 

Polycations such as protamine or streptomycin may also be used for differential protein precipitation. These materials bind to negatively charged compounds and hence neutralize a large proportion of the charge they possess. Polycations can be used most advantageously if the desired protein is not precipitated by them. As shown in Table 10-3, PEP carboxykinase from R. rubrum remains soluble while three-quarters of the undesired proteins precipitate with protamine sulfate. On the other hand, the fact that polycations irreversibly remove many anionic proteins somewhat limits their use. When polycations are used they must be... 

Recently, automated sample preparation approaches utilizing parallel sample processing have been described for SPE [10], LLE [9], simple dilutions [11], and protein precipitation [8]. These procedures utilize commercially available workstations for liquid handling in a 96-well multichannel plate format. These workstations are evolving rapidly and are constandy gaining additional capabilities. A recent article has reviewed the most common types and describes the major advantages of each [36]. 

The three main formats for sample preparation used in drug-discovery are protein precipitation (PPT), SPE, and LLE. Several examples of off-line sample preparation have been reported and involve SPE [37,38,46,47], LLE [38,48], and PPT [39,49]. In each of the examples cited, semi- or fully automated strategies for liquid handling were incorporated to enhance throughput. Even with the recent popularity of on-line methods, off-line techniques continue to be widely employed. The key advantage to off-line methods is that sample preparation may be independently optimized from the mass spectrometer and does not contribute overhead to the LC-MS injection duty cycle. 

A Unal example of direct bioanalysis was recently published by Dethy et al. and involves the appUcation of infusion nanoelectrospray (nano-ESI) from a silicon chip [110]. In this example, supernatant obtained from protein precipitation was directly infused with an automated pipette-tip delivery system. Individual, conductive pipette tips that contain sample were sequentially introduced to the backplane of a silicon chip for analysis. The front plane of the chip that consisted of 100 individual nano-ESI nozzles, was positioned near the API orifice of a TQMS for direct serial analysis. Quantitation of verapamil and its metaboUte norverapamil occurred in human plasma over a range of 5-1000 ng/mL. It is possible to achieve analysis times of less than 1 minute per sample with this technology. An important advantage, demonstrated by this work, is the unique abiUty to avoid system carryover with this device [110]. 

The only disadvantage of acetone containing extracting solvents is that even traces of acetone will interfere with protein precipitation assays [86]. The advantage of acetone compared to methanol (or ethanol) has been pointed out since the early 1980 s [87] and has been demonstrated for a variety of different matrices in the past twenty years. Astonishingly, aqueous methanol is still used more frequently than aqueous acetone in the extraction of procyanidins. 

Earlier FAAS techniques for measuring serum copper levels which included protein precipitation with trichloroacetic acid (Olson and Hamlin, 1968) and/or solvent extraction have been superseded by simpler procedures using either large sample dilution or viscosity adjusted reference solutions with minimal dilution. The analyses are made mainly using continuous nebulization, discrete sample injection or by flow injection techniques with little advantage gained from flame adaptors to increase sensitivity. 

The determination of nitrite in egg highlights the beneficial aspects of sonoelec-trochemical analysis in difficult matrices. Moreover, the advantages of the technique include significant simplification over the liquid chromatographic procedure currently employed for nitrite detection [69], which involves hot water extraction, protein precipitation and solid-phase extraction / purification. 

The micellar solvent system used in this assay has several advantages, in addition to rapid separation SDS is an effective-denaturing agent and, as such, can be used to stop reactions without the need for protein precipitation with trichloroacetic acid or heating. Since SDS also has a unique solubilizing power, it can be used for direct injection of concentrated protein solutions into the RPLC system, without time-consuming steps to remove protein precipitates or extraction of folate analogues. Therefore, the assay is simple, rapid, inexpensive, and applicable to crude hydrolase preparations. 

Specific methods Immunochemical methods have been developed for identification and specific determination of proteins that have no catalytic activity. The method is based on the special relationship between a protein and a specific antibody that has been raised against the protein. The advantages of an immunoassay lie in its high specificity and detectability. Since the specificity is independent of the activity of the antigen protein, the method measures all enzyme molecules, inactive as well as active. The methods depend on the availability of specific antisera directed against the particular enzymes under study. The antibody is raised in an experimental animal by injection of a sample of protein antigen. Selective precipitation method and enzyme linked immunosorbent assay (ELISA) are commonly used for the specific detection and reliable measurement of a particular enzyme in a raw sample, such as a tissue extract or body fluids. 

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