Protein ammonium sulphate precipitation

Figure 5. Analytical isoelectric focusing. Ultrathin layers (0.4 nun) of polyacrylamide with ampholytes pH 2-11 were used. Samples of 10 pg of protein in 10 pi of 1 % glycine were applied. A.- Silver staining. B.- Stain for activity on overlays containing pectin in tris/HCl buffer at pH 8.0 with CaClj M.- Broad pi Calibration Kit protein (Pharmacia), samples of 5 pg of protein were applied. 1.-Ammonium sulphate precipitated proteins from cultures on pectin. 2.- Fractions with PNL activity eluted from the Superdex 75HR1030 column. 3.- Purified PNL.

Figure 8.14. Production of recombinant human growth hormone (rhGH) in E. coli (as an intracellular protein). Subsequent to fermentation, the cells are collected by centrifugation or filtration. After homogenization, nucleic acids and some membrane constituents are precipitated by the addition of polyethyleneimine. Ammonium sulphate precipitation of the supernatant concentrates the crude rhGH preparation. Chromatographic purification follows, as illustrated...

There is frequently a need to change buffer conditions during protein preparations a typical example is when an ammonium sulphate precipitation step is followed by ion-exchange chromatography. The ammonium sulphate can be removed from the re-dissolved pellet by prolonged dialysis, but it is preferable, for reasons of speed and stability, to pass the solution over a Sephadex G-25 coarse column, preequilibrated with the start buffer for the next column for such coarse operations the sample volume can amount to ca. 15-20 % of the column volume. 

The preparation of the cyclic AMP binding protein from bovine muscle has been described [150]. It involves homogenisation, centrifugation, pH 4.8 precipitation and ammonium sulphate precipitation, followed by fractionation on DEAE cellulose. The preparation binds 0.3 pmol of cyclic AMP per ixg of protein and has an enzymatic activity of 24 pmol of P per jxg of protein per 10 min. Over 200 /ig of the protein can be quantitatively adsorbed on a single Millipore filter. The yield of binding protein from 500 to 1000 g of muscle is sufficient for more than 100000 assay tubes. The binding activity is stable for 18 months at -20°C. 

The cyclic GMP binding protein is prepared from lobster tail through the dialysed ammonium sulphate precipitation step described by Kuo and Greengard [76]. The binding activity is stable for months at -70 C, but activity is lost with repeated thawing and freezing. 

Precipitated Protein.—Ammonium sulphate, when added to saturation, precipitates from solution all proteins and derived proteins except peptones. Sodium chloride similarly precipitates only glohulins if the solution is neutral, but all ex -ept peptonesj if acid is added. 

Used as cmde extract.Partially enriched by heat treatment of cmde extract at 67 °C for 30 min, ammonium sulphate precipitation (45 % saturation), resolubilisation of the precipitated protein in 50 mM TEA-maleate buffer, pH 7.5, and chromatography on a Sepharose G 200 column. 

When, however, 13(S)-HPOD was incubated with an ammonium sulphate precipitate of defatted com germ, the resulting 12-oxo-PDA was not optically pure but a mixture of enantiomers in a ratio of 82.18 [35]. The 12-oxo-PDA obtained by incubation of 13(S)-HPOT with flaxseed extracts was found to be a racemic mixture [36]. These results may be due to low or absent allene oxide cyclase activity in the preparations used [34]. The allene oxide cyclase, a novel enzyme in the metabolism of oxygenated fatty acids was partially characterised and found to be a soluble protein with an apparent molecular weight of about 45 kDa which catalysed specifically the conversion of allene oxide into 12-oxo-PDA [34]. The allene oxide was very unstable and was rapidly hydrolysed in aqueous media into stable 12-oxo-13-hydroxy-9(z),15(z)-octadecadienoic acid (a-ketols) [34]. 

Whole serum was used for immuno-electrophoresis studies and measurement of serum thyroxine and protein-bound iodine but a crude -globulin preparation was obtained by ammonium sulphate precipitation (4) to Inject into mice for bioassay purposes this was... 

A two-step purification procedure was used for the isolation of lethal protein toxin, Toxin-PC, including 50% ammonium sulphate precipitation followed by DEAE-ion exchange chromatography. Toxin-PC was homogenous in polyacrylamide gel electrophoresis with a SDS-molecular weight of near 15 kDa and devoid of glycoprotein moiety. As described in the results section, lethal Toxin-PC was highly active on isolated heart and nerve... 

Enzymes are proteins of high molecular weight, several of which have been isolated in a pure State consequently their precise nature is in some instances still obscure. They form solutions in water and in dilute salt solutions, and are precipitated when such solutions are saturated with ammonium sulphate. 

Extracellular Enzyme Activities. The protein, carrier of the polygalacturonase and pectinmethylesterase activities, was salted out from the H.annuus 1805 culture medium by adding ammonium sulphate to 70 % saturation. The precipitate was separated by centrifugation (30 min, 4500 xg), diluted with 0.1 M phosphate buffer (pH 7.0) and dialyzed for 12 hours against distilled water. After dilution to the required volume with the same buffer, both enzyme activities under study were determined in the solution. 

Separation of bound and free analyte. This is often done by adsorption of the free analyte on activated charcoal or precipitation of the protein bound fraction with ammonium sulphate. 

Some separation techniques rely on the physical removal of one of the fractions charcoal will strongly adsorb the free fraction allowing its ready removal by centrifugation the addition of dextran reduces the tendency of charcoal to strip bound antigen from the complex alternatively, the bound fraction may be precipitated by the addition of suitable concentrations of various protein precipitants such as alcohol, ammonium sulphate and polyethylene glycol (PEG). 

Many cytosolic proteins are water soluble and their solubility is a function of the ionic strength and pH of the solution. The commonly used salt for this purpose is Ammonium Sulphate, due to its high solubility even at lower temperatures. Proteins in aqueous solutions are heavily hydrated, and with the addition of salt, the water molecules become more attracted to the salt than to the protein due to the higher charge. This competition for hydration is usually more favorable towards the salt, which leads to interaction between the proteins, resulting in aggregation and finally precipitation. The precipitate can then be collected by centrifugation and the protein pellet is re-dissolved in a low salt buffer. Since different proteins have distinct characteristics, it is often the case that they precipitate (or salt out ) at a particular concentration of salt. 

Table 15.3 Crystallization conditions for DNA-protein complexes using ammonium sulphate as a precipitant... 

Seeds were grounded and soaked in 20 mM Tris-HCI buffer (pH 7.6) at 4°C for 24 h. The seeds were blended in a blender to extract the proteins followed by centrifugation (30,000g) at 4°C. Then 450 g/l of ammonium sulphate were added to the supernatant to 70% saturation. The precipitate was removed by centrifugation and the supernatant was extensively dialysed against distiled water. The dialysed protein extract was freeze dried and used for chromatographic separation. 

---