Precipitation, protein

Protein precipitation is a technique used in toxicology to remove the protein content of human body fluids and tissues before they are analysed. The reason for this is that these samples of protein content can vary from 6% to more than 50%, by weight, in some tissues. This can greatly affect the possibility of detecting and quantifying drug and metaboUte concentrations. 

Generally, a precipitation reagent, such as an organic solvent (e.g., acetonitrile or methanol) or a salt and an acid (e.g., ammonium sulphate and hydrochloric acid), will be used. Once the proteins have been precipitated, the solid protein will then be removed by filtering or by centrifugation. The rest of the liquid sample will then be further cleaned up before extraction, or extraction will occur immediately after the protein precipitation stage. 

Endo nous and exr enous substances often need to be determined in samples, such as plasma and serum, which are very protein-rich and complex matrices. The protein content usually makes the determination difficult and protein removal is often required, for example, by protein predpitation. Most used protein predpitation agents are organic solvents, salts, and adds. Table 9.7 shows some of the most used predpitants and the amount needed to predpitate 95 and 99% of all proteins. The precipitated proteins are removed by centrifugation, and the supernatant is used for analysis. 

When miscible organic solvents are added to aqueous solutions, the dielectric constant (e°) is decreased. This leads to compressed hydrated layers of the proteins, which in its turn allows proteins to interact with each other. The lower the dielectric constant, the more easily this interaction occurs, causing aggregation and predpitation. 

Add 5 ml chloroform isoamyl alcohol (24 1) (to eliminate phenols). Vortex, centrifuge on high for 10 min. Save supernatant for step 14. 

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Brucke s reagent (protein precipitant) dissolve 50 g of K1 in 500 mL of water, saturate with Hglj (about 120 g), and dilute to 1 liter. 

The protein precipitate is washed with water, redispersed at pH 7, and then spray dried. Typical commercial soy protein isolates contain greater than 90% cmde protein, dry wt basis. 

Factor VIII, immunoglobulin, and albumin are all held as protein precipitates, the first as cryoprecipitate and the others as the Cohn fractions FI + II + III (or FII + III) and FIV + V (or FV), respectively (Table 7, Fig. 2). Similarly, Fractions FIVj + FIV can provide an intermediate product for the preparation of antithrombin III and a-1-proteinase inhibitor. This abiUty to reduce plasma to a number of compact, stable, intermediate products, together with the bacteriacidal properties of cold-ethanol, are the principal reasons these methods are stiU used industrially. 

Silver nitrate is astringent and a protein precipitant, which is not medically desirable. Other forms of silver have been used to avoid this problem, including coUoidal silver, silver-protein preparations, and finely divided silver metal called Katadyn silver. 

Water-soluble polymers and polyelectrolytes (e.g., polyethylene glycol, polyethylene imine polyacrylic acid) have been used success-hilly in protein precipitations, and there has been some success in affinity precipitations wherein appropriate ligands attached to polymers can couple with the target proteins to enhance their aggregation. Protein precipitation can also be achieved using pH adjustment, since proteins generally exhibit their lowest solubility at their isoelectric point. Temperature variations at constant salt concentration allow for frac tional precipitation of proteins. 

Purification of photoprotein. The dialyzed photoprotein solution was centrifuged to remove precipitates, and then subjected to fractional precipitation by ammonium sulfate, taking a fraction precipitated between 30% and 50% saturation. The protein precipitate was dissolved in 50 ml of 10 mM sodium phosphate, pH 6.0, containing 0.1 mM oxine ( pH 6.0 buffer ), dialyzed against the same buffer, and the dialyzed solution was adsorbed on a column of DEAE-cellulose (2.5 x 13 cm) prepared with the pH 6.0 buffer. The elution was done by a stepwise increase of NaCl concentration. The photoprotein was eluted at 0.2-0.25 M NaCl and a cloudy substance (cofactor 1) was eluted at about 0.5 M NaCl. The photoprotein fraction was further purified on a column of Sephadex G-200 or Ultrogel AcA 34 (1.6 x 80 cm) using the pH 6.0 buffer that contained 0.5 M NaCl. 

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). 

Fig. 1. Relationship between reactor mean velocity gradient and particle size for isoelectric soya protein precipitate. Open symbols represent precipitate diameter for 50% oversize Closed symbols represent precipitate diameter for 90% oversize [51]...

Fig. 2. Flow of protein precipitates through various pumps [107]...

Protein precipitate, insect cells Hvbridoma. Mammalian cells... 

Protein precipitate, hybridoma cells Insect cells Mammalian Cells... 

A similar approach has been suggested in other studies of plant cells [57] and protein precipitates [133]. However, information on the rate of the size distribution shift process cannot be inferred from chain-length measurements made only at the beginning and end of the experiment. To date, there have been no reports on the progressive modification of the size distribution of plant cells subjected to continued exposure to turbulent forces. There are, however, a number of studies which address the break-up of mycelial hyphae in agitated vessels... 

Many use the hexokinase procedure without protein precipitation. That this is a procedure which is not acceptable is illustrated in Table II where it is shown that unless hemoglobin is removed there will be interference in the results obtained. In severely hemolyzed blood, errors as high as 25% are not uncommon. 

If the fluorometric procedure is used, with protein precipitation, then bilirubin will not interfere with the hexokinase procedure. Even if protein precipitation is not resorted to, the interference from bilirubin does not become significant until one is dealing with an infant in severe jaundice. This can be seen in Table III. 

If the fluorometric method is used after protein precipitation then the glucose can be readily assayed on 1 pi of plasma with the hexokinase procedure. 

Effect of hemolysis on the assay of glucose by the fluorescence-hexokinase method without protein precipitation. 

Effect of bilirubin on the glucose fluorometric hexokinase procedure with and without protein precipitation, ... 

Samples with higher protein levels (yogurts), are initially treated with hydrochloric acid and after protein precipitation the supernatant is filtered and injected into the HPLC column. The separations performed with a LiChroCART RP18 column used a mixture of acetonitrile and formic acid as the mobile phase. A baseline quantification of the carminic acid was possible in the presence of other coloring agents, with excellent recuperation, selectivity, accuracy, and precision. ... 

Kakizuka A. Protein precipitation a common etiology in neurodegener-ative disorders Trends Genet 1998 14 396 -02. 

Add a quantity of BMPA to the protein solution to obtain at least a 5-fold molar excess of maleimide reagent over the amount of thiol present in the protein. The final concentration of organic solvent in the protein solution should not exceed 10 percent to prevent protein precipitation. Mix thoroughly to dissolve. 

DPDPB is insoluble in aqueous solutions and should be initially dissolved in an organic solvent prior to addition of a small aliquot to a buffered reaction medium. Preparation of a stock solution in DMSO at a concentration of 25 mM DPDPB works well. The addition of an aliquot of this stock solution to the conjugation reaction should not result in more than about 10 percent organic solvent by volume in the buffered mixture or protein precipitation may occur. 

SADP is hydrophobic and should be dissolved in organic solvent prior to addition of a small aliquot to an aqueous reaction. Concentrated stock solutions can be prepared in dry DMSO or DMF. Final concentration of the organic solvent in a crosslinking reaction should not exceed about 10 percent to prevent protein precipitation or denaturation. 

Fluorescein-5-maleimide is slighdy soluble in aqueous solutions above pH 6 ( lmM concentration). It may be dissolved in DMF at higher concentrations and a small addition of this solution made to an aqueous reaction mixture to initiate labeling. Do not exceed 10 percent DMF in the reaction buffer to avoid protein precipitation. At pH 8, the reagent has an extinction coefficient at 490nm of about 78,000M 1 cm-1. 

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