Protein precipitation efficiency

Comparison of Protein Precipitation Efficiencies of Precipitants upon Treatment with Human Plasma... 

Relative extraction efficiencies of polar polymeric neutral, cation, and anion exchange sorbents (HLB, MCX, and MAX) for 11 beta antagonists and 6 beta agonists in human whole blood were probed.109 Initial characterization of MCX and MAX for acidic and basic load conditions, respectively, showed that both the agonists and antagonists were well retained on MCX, while they were recovered from MAX in the wash with either methanol or 2% ammonia in methanol (see Table 1.6). Blood samples were treated with ethanol containing 10% zinc sulfate to precipitate proteins and the supernatants loaded in 2% aqueous ammonium hydroxide onto the sorbents. After a 30% methanol and 2% aqueous ammonia wash, the analytes were eluted with methanol (HLB), 2% ammonia in methanol (MCX), or 2% formic acid in methanol (MAX). The best recoveries were observed with MCX under aqueous conditions or blood supernatant (after protein precipitation) spiked sample load conditions (see Table 1.7). Ion suppression studies by post-column infusion showed no suppression for propranolol and terbutaline with MCX, while HLB and MAX exhibited suppression (see Figure 1.6). 

Extraction efficiency. Recovery of the analyte from biological matrix after sample pretreatment (i.e., liquid-liquid extraction, solid-phase extraction, protein precipitation, etc.) to remove endogenous substances. 

For the purpose of achieving a selective SPE extraction that eliminates matrix components to the greatest extent possible without loss of analytes, an extensive optimization of the wash/elute conditions was conducted, the procedure is similar as described in previous examples. In order to achieve high efficiencies of extraction and reproducibility of quantification, a protein precipitation with ACN/methanol prior to SPE extraction is found important to assist releasing VitD metabolites from serum protein and derivatization by PTAD plays a critical role to improve the stability of VitD metabolites. 

It is not possible, currently, to offer an explanation for these observations, but perhaps it is permissible to speculate. The low efficiency with methylene chloride might be explained by the fact that proteins precipitate when plasma is extracted with this solvent. Since A9-THC is bound to protein, it may co-precipitate and not be extracted from precipitate. However, co-precipitation with proteins cannot be invoked to explain the results with diethyl ether since this solvent does not cause protein precipitation. Furthermore, it is also necessary to explain the observation on extraction with toluene and the differences between rabbit and human plasma. The coprecipitation argument is simply not applicable to these phenomena. 

The use of methanol and ethanol, two solvents that can be well mixed with water, should be discussed separately, as they are used for several types of sample preparation on the basis of similar expected effects, but for different purposes. The feature common to both solvents is the observation that most of the sample proteins precipitate when the concentration of these compounds exceeds ca. 40 percent (v/v), thus enabling the analyst to separate the protein fraction by centrifugation or microfiltration. This way, either the proteins not intended for further analysis (e.g., enzymes or proteins that were inadequately hydrolyzed) can be removed [62], or, the purification of Se-containing proteins by successive solvent extractions can be achieved [12]. At concentrations of less than 40 percent (v/v), both methanol and ethanol are usually mixed with 0.1 moll-1 HC1 however, the use of these mixtures entails a relatively low extraction efficiency of Se (10-14 percent). Therefore, they are intended only for the extraction of water-soluble Se species, generally free selenoamino acids [15, 21, 63, 64]. 

Certain techniques are very good at particular tasks (such as protein precipitation for removal of proteins and cellular components), but perhaps not as good in general. With the emphasis on greater efficiency, many of these approaches have become automated or semiautomated. Thus, there has been greater emphasis on the 96-well formats an approach that can lend itself more readily to automated workstations. This format has been used successfully for protein precipitation, liquid—liquid extraction, and solid-phase extraction [8-10]. Ultrafiltration (UF) in a 96-well format is also being evaluated and shows some potential, but products and applications are not yet fully developed. Automated techniques for sample preparation and each of the sample preparation techniques listed in Table 1 are described below. 

One especially facile workstation, the Quadra 96 (Tomtec), has proven to be a highly efficient 96-well liquid handler for SPE, LLE, and protein precipitation. Using this device as a liquid handler, aliquots of solvents can be manipulated and transferred, in parallel, to each of the wells in the plate in less than 1 min. One disadvantage of this device is the requirement for samples to be in a 96-well format at the start of the procedure. Once this requirement is met, however, and samples are in the correct format, parallel processing can proceed quickly in a semiautomated format, as shown in Fig. 8. 

Protein precipitation using a change in pH is usually successful for concentrated systems. It may not be efficient for low concentrations however. In general, protein solubility rises with increasing salt concentration up to 0.2M. This process is known as salting in. Under... 

Solution dH Several investigators (2, 12, 13) have indicated that the solution pH is an important determinant in the precipitation efficiency, and the optimum pH level will vary with both the protein (12) and the polyelectrolyte (5). However, the optimum pH for precipitation of protein by CMC did not change with the degree of substitution (DOS) of the CMC (12). This dependence is expected for the formation of an electrostatic complex. Changes in pH will affect the charge on the polyelectrolyte and the charge distribution on the protein. 

Ionic Strengths If the protein-polymer complex is formed as a result of electrostatic interactions, increased ionic strength should serve to reduce the attraction between the oppositely charged macromolecules, and decrease the precipitation efficiency. This is observed at pH 4.2 in Figures 3 and 4 for lysozyme and ovalbumin, respectively, and in Figure 5 for lysozyme at pH 5.8 and 7.5. 

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