Protein precipitants, types

Filter the solution through a 0.2 pm filter (low protein binding type) to remove any precipitated protein or particles. 

Direct injection of plasma or supernatant after protein precipitation on a short column with a high liquid flow rate is a common method for reducing analysis time in the pharmaceutical industry. The direct injection of a sample matrix is also known as the dilute-and-shoot (DAS) approach.62 DAS can be applied to all types of matrices and approaches and is the simplest sample preparation method with matrix dependency. Direct injection can also be approached through the extraction of eluent from PPT, SPE, and LLE onto a normal phase analytical column. The procedure is called hydrophilic interaction liquid chromatography (HILIC)70110111 and it avoids the evaporation and reconstitution steps that may cause loss of samples from heat degradation and absorption. 

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]. 

To assess Se distribution and speciation in Brazil and other types of nuts, the defatted and powdered samples were fractionated into proteins and cytosol as shown by a Bow chart in Figure 21.1 [13, 14], For the evaluation of Se binding to proteins by SEC, protein precipitate was solubilized with SDS-containing acetate buffer. For the speciation of low-molecular-weight (LMW) Se compounds released from proteins, two procedures of protein hydrolysis were applied and the results obtained were compared. It was shown that acid hydrolysis with methane-sulfonic acid allowed for more efferent release of selenomethionine from proteins as compared with enzymatic proteolysis [14],... 

A number of reports in the literature indicate that matrix effect is dependent on ionization type, sample preparation, and bio-fluid type [104,108,114, 116-119]. In 2003, Dams et al. [119] demonstrated that the APCI was less susceptible to matrix effect, thereby allowing for simplification of sample preparation prior to LC/MS/MS analysis without jeopardizing the quality of quantitative data, as shown in Table 7-8. More recently, Souverain et al. [117] investigated the matrix effect in bio-analysis of illicit drugs with both LC/ESI-MS/MS and LC/APCI-MS/MS. Four procedures—liquid-liquid extraction (LLE), SPE, protein precipitation (PP) with acetonitrile (ACN), and protein precipitation with perchloric acid (PA)— were tested to evaluate... 

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]. 

Six published and personal examples have been detected of Bence Jones protein precipitating in the cold, but all under 20°C, and causing no symptoms in the patients. They were mostly type L, and this class shows an excess among monoclonal cryoimmunoglobulins. Rarely, true crystals may form in the cold, and any such protein would be welcome to X-ray crystallographers. 

Among the methods of purifying protein, precipitation is the most useful and typical for both small and large scale procedures. The precipitation methods are classified into 4 types, salting-out, organic solvent precipitation, pH changing precipitation and water-soluble precipitation. The precipitation is usually carried out early and the total protein concentration should be >0.1 mg/mL. 

In order to distinguish between nonpro-tein-bound platinum and unbound cis-Pt or carbo-Pt, trichloroacetic acid (TCA) protein precipitation, or the precipitation of plasma proteins with cold ethanol and ultrafiltration, can be used (Ma et al. 1996). Other batch-type experiments were reported to study the binding behavior of Pt-compounds using ET-AAS as the determination method (Parsons et al. 2003). Gel-permeation chromatography was used to study Pt species in extracts from plants treated with Pt-salts (Messerschmidt et al. 1994), while Alt et al. (2002) studied the bonding stage of Pd in endive. 

In general, the larger the quantity of proteins added, the more tannins are eliminated. However, the reaction depends on the type of proteins, and no direct correlation has been observed between the quantity of protein added and the quantity of tannin eliminated (Table 10.4). Turbidity (Siebert et al., 1996), as well as the type and quantity of tannin-protein precipitates, depend on the relative concentrations of the various components (Calderon et al., 1968). 

Defatted oil seed meals are known to contain large amounts of nutritional proteins which can be recovered for use in food and pharmaceutical industries. Protein recovery from solution is often achieved through precipitation followed by solids-liquid separation. For efficient recovery, it is imperative to maximize the solids yield and the mean particle size, and minimize the spread of particle size distribution (PSD). In this research program, recovery of canola and sunflower protein precipitates has been investigated. Three different types of precipitators (batch, MSMPR, and tubular) and four different precipitants (HCl, carboxymethylcellulose (CMC), sodium... 

Experiments were performed to determine the optimum pH for the extraction of proteins from the industrially defatted canola meal (Federated Co-op Ltd., Saskatoon, SK) and sunflower meal (Cargill Inc., West Fargo, ND), and the minimum solubility pH (isoelectric pH) for precipitation. Details of the experimental methods can be found in (7) and (13) respectively. The extracted protein solutions were then used to recover proteins by precipitation. Four different types of precipitants aqueous HCl, HMP, CMC, and ammonium sulphate, were used for studies on the yields of canola protein. Further studies on isoelectric precipitation (using aqueous HCl as a precipitant) were carried out in three types of precipitators (Batch, MSMPR, and tubular) to study kinetic parameters and effects of precipitator type and operating conditions on PSD. The first two types of precipitators were used for canola protein and all three types were used for sunflower protein. Details of these experimental setups can be found in (7) for canola protein and (13,11) for sunflower protein. 

This type of chiral stationary, however, proved to be very effective. The separation of the enantiomers of hexabarbital on this stationary phase by the direct injection of blood serum is shown in figure 11.27. Chromatogram A was obtained after 20 injections of serum and chromatogram B after 60 consecutive injections of blood serum. It is seen that here is very little column deterioration and that, although the tail of the major peak has become a little extended after 60 injections, the column could still be used very effectively for the analysis (it should be noted, that it is essential to employ titanium frits in the column, to prevent protein precipitation, which could occur on stainless steel frits). 

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