Protein precipitation purpose

From animal tissue, especially bovine lung and liver (e. g. autolysis of comminuted tissue parts, heating with ammonium sulfate in alkaline solution, filtration and acidification yield heparin as complex with protein, removal of fat with alcohol and treatment with trypsine for the purpose of decomposition of proteins, precipitation with alcohol and various purification methods). 

With the use of the inorganic sulfates or chlorides, protein precipitation does occur but the process is reversible. This does not occur when acids or organic solvents are used for protein precipitation, since the biological activity of the proteins in this case is irreversibly destroyed. On the other hand, use of mineral or organic acids, although valuable for deproteinization purposes, has raised prob-... 

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. 

Sample Preparation/Extraction The process of separating potentially interfering components from a sample prior to LC-MS analysis for the purposes of improving sensitivity, specificity, and/or method ruggedness. Variations include solid phase extraction (SPE), liquid-liquid extraction (LLE), and protein precipitation (PPT). Extraction may be performed off-line, in which the cleanup is completely independent from the LC-MS analysis, or on-line, in which the cleanup is integrated directly into the LC-MS analysis. 

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. 

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]. 

Resuspend the precipitated protein obtained from step 10-31 in a second 10 ml cold water. Remove the undissolved protein by centrifugation and combine this supernatant solution with that obtained in step 10-31. This is supernatant V. Note its volume and remove a 0.5 ml sample for assay purposes. The insoluble protein precipitate may be discarded. 10-33. Cut a convenient length (15 to 18 in.) of cellulose dialysis tubing (jin. diameter). Wet the tubing with distilled water and tie two knots in one end of it, as shown in Figure 10-7. 

For preparative purposes, it is perhaps more convenient to start from pancreas acetone powder (pancreatin). Ninty-five per cent pure bovine procarboxypeptidase A has been obtained by ammonium sulfate fractionation of pancreatin extracts and isoelectric precipitations (47). When the proteins precipitated by 0.39 saturated ammonium sulfate are chromatographed on DEAE-cellulose in a concentration gradient of pH 8.0 phosphate buffer, the two last and most acidic peaks contain procarboxypeptidase A in an electrophoretically homogeneous form (48). The molecular weight of the protein determined by light scattering and sedimentation-diffusion is 94-96,000. Its isoelectric point in univalent buffers of 0.2 ionic strength is below 4.5. 

Prompt stabilization of ascorbic acid is especially important in the case of plasma or serum samples. Metaphosphoric acid is often used for this purpose because it also serves as a protein precipitant. Such properties are desirable in the inactivation of oxidase and the catalytic eflFect of copper. Oxalic acid is an attractive stabilizer for ascorbic acid analysis because of its lower cost and greater stability however, it is not a protein precipitant, therefore, it has a limited use for the extraction of animal tissues. The use of ethylenediaminetetraacetic acid (EDTA) in addition to the metaphosphoric acid has been recommended (96). EDTA would chelate divalent cations, and a study has shown it will stabilize ascorbic acid in the presence of copper for several days (96). Perchloric acid has been used also but because of its inherent dangerous properties its use is generally avoided. Trichloroacetic acid and EDTA also seem appropriate extractants for ascorbate in plant materials (97). 

Protein solubility is a thermodynamic characteristic of the protein/solvent system dehned as the concentration of soluble protein in equilibrium with the solid phase at a given pH, temperature, and solvent composition (Flynn, 1984 Arakawa and Timashelf, 1985 Middaugh and Volkin, 1992). For practical purposes, solubility of proteins can be dehned as the maximum amount of protein that remains in a visibly clear solution (i.e. does not show protein precipitates, crystals, gels, or hazy soluble aggregates), or does not sediment at 30,000 g centrifugation for 30 min (Schein, 1990 Ducruix and Reis-Kautt, 1990). 

In the presence of CH2-H4folate, FdUMP forms a specific, stable complex with thymidylate synthetase in which all components are covalently bound as depicted in Fig. 1. The affinity constant of this complex is suflSciently high that, with typical concentrations of components used in most experiments, the limiting reagent (FdUMP or enzyme) is completely bound. Using [ H]FdUMP of high specific activity, low levels of complexes present in solution may conveniently he assayed by retention on nitrocellulose filter membranes under conditions in which the free nucleotide is readily removed. The radioactivity remaining on the filter is determined to quantitate the complex. Other conventional methods (e.g., gel filtration, charcoal adsorption of free FdUMP, protein precipitation) may be used for this purpose, but are more tedious and apparently less eflicient. There are expectedly few proteins that will form isolable... 

When the activity and other properties of the several times recrystallized new enzyme protein are compared with those of the uncrystallized precipitate obtained in the first stages of the process, it Is found that even in the first stages, the enzyme is present in sufficiently pure form for most purposes. 

In the particular example shown, zinc sulfate and barium hydroxide are being dispensed into the test tube so as to precipitate the proteins. The filtrate obtained is the filtrate from 10 microliters of serum. This can be used for several purposes and in the application being referred to, an amount equivalent to 3 microliters is being used for sugar determination, by the hexokinase procedure and an amount equivalent to 3 microliters is being used for urea estimation with diacetylmonoxime (15). 

Polyphenols are ubiquitous in all plant organs where they are found as monomers or in polymerised forms (Schofield et al, 2001). In addition to the beneficial effect of poljq)henols, they also bind minerals and precipitate proteins and carbohydrates, in effect reducing the nutritive value of foods. Polyphenols have been classified for nutritional purposes into extractable and non-extractable types (Bravo, 1998). Extractable polyphenols are low-and intermediate-weight phenolics while non-extractable polyphenols have high molecular weight and are insoluble in normal solvents. 

As with urine, saliva (spumm) is easy to collect. The levels of protein and lipids in saliva or spumm are low (compared to blood samples). These matrices are viscous, which is why extraction efficiency of xenobioties amoimts to only 5 to 9%. By acidifying the samples, extraction efficiencies are improved as the samples are clarified, and proteinaceous material and cellular debris are precipitated and removed. Some xenobioties and their metabohtes are expressed in hair. Hair is an ideal matrix for extraction of analytes to nonpolar phases, especially when the parent xenobioties are extensively metabolized and often nondetectable in other tissues (parent molecules of xenobioties are usually less polar than metabolites). Hair is a popular target for forensic purposes and to monitor drug compliance and abuse. Human milk may be an indicator of exposure of a newborn to compounds to which the mother has been previously exposed. The main components of human milk are water (88%), proteins (3%), lipids (3%), and carbohydrates in the form of lactose (6%). At present, increasing attention is devoted to the determination of xenobioties in breath. This matrix, however, contains only volatile substances, whose analysis is not related to PLC applications. 

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