Ammonium sulfate fractionation

All of the following steps should be performed at 4 °C unless otherwise stated. 

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Cathepsin D (from bovine spleen) [9025-26-7] Mr 56,000, [EC 3.4.23.5]. Purified on a CM column after ammonium sulfate fractionation and dialysis, then starch-gel electrophoresis and by ullracentrifugal analysis. Finally chromatographed on a DEAE column [Press et al. Biochem J 74 501 I960],... 

Thirty-two years later, Ko jima etal. (2000a) purified Latia luciferase by the following steps ammonium sulfate fractionation, gel-filtration, affinity chromatography and Mono-Q anion-exchange FPLC. 

Cormier and Dure (1963) found another type of luciferin and called it protein-free luciferin. Protein-free luciferin was found in the vapor condensate of freeze-drying whole animals, and also in the 3 5-56 % ammonium sulfate fraction of the crude extract noted above. The protein-free luciferin behaved like an aromatic or heterocyclic compound and it was strongly adsorbed onto Sephadex and other chromatography media, requiring a considerable amount of solvent to elute it. The luminescence reaction of protein-free luciferin in the presence of luciferase required a 500-times higher concentration of H2O2 compared with the standard luciferin preparation. Both types of the luciferin preparation had a strong odor of iodoform. 

A wheat germ, cell-free, translation extract was fractionated into three concentrated parts using ammonium sulfate the 0 - 40 % saturated fraction, the 40 - 60 % saturated fraction, and the ribosome fraction. These fractions were tested for their ability to enhance the translational activity of the wheat germ, cell-free extract for dihydrofolate reductase. The fortified cell-free system supplemented with the 0 - 40 % ammonium sulfate fraction enhanced the efficiency of protein synthesis by 50 %. 

Previously, it has been reported that the amounts of eukaryotic initiation factors in wheat germ extract prepared by a common method were deficient for the translation of some kinds of mRNAs including a-amylase mRNA and (i-globin mRNA [2]. Therefore, it can be expected that the activity of wheat germ extract prepared by a common method can be enhanced by the simple addition of extract containing deficient initiation factors. In this study, a wheat germ extract was further purified partially by ammonium sulfate fractionation... 

Fig. 1. Reconstruction of the cell-free protein synthesizing system with the partially purified wheat germ extracts. Control normal wheat germ cell-free system, (I) 0 - 40 % ammonium sulfate fraction 3 pi, 40 - 60 % ammonium sulfate fraction 4 pi, and ribosome 3 pi were added to 25 pi reaction mixture, (II) 0-40 % ammonium sulfate fraction 4 pi, 40 - 60 % ammonium sulfate fraction 4 pi, and ribosome 1.5 pi were added to 25 pi reaction mixture.

Preparation of 0 - 40 % and 40 - 60 % ammonium sulfate fractions from S170 supernatant... 

The SI70 supernatant (220 ml) was made to 40 % saturation with solid ammonium sulfate, stirred for 20 min, and then the precipitate was collected by centrifugation at 15,000 g for 15 min. The precipitate was suspended in small volume of buffer B-50 at pH 7.6 containing 20 mM HEPES/KOH, 0.1 mM EDTA, 1 mM dithiothreitol, 10 % (v/v) glycerol, and 50 mM potassium acetate. The 60 % saturated ammonium sulfate solution was prepared similarly. Protein concentrations for 0 - 40 % and 40 - 60 % ammonium sulfate fractions were 4.2 mg/ml and 4.7 mg/ml, respectively. 

The catalytic activities of the fortified wheat germ cell-free systems supplemented with each fraction were investigated (Fig. 2). As shown in Fig. 2, only 0 - 40 % ammonium sulfate fraction showed an enhancement in DHFR protein synthesis. This enhancement of protein experimental results and the fact that the various eukaryotic initiation factors are contained in synthesis was also confirmed by SDS-PAGE and autoradiography (Fig. 3). From the above 0-40 % ammonium sulfate fraction [5, 6], it can be concluded that the amount of initiation factors in a conventionally prepared wheat germ cell-fi extract is deficient for the translation of DHFR with internal ribosome entry site. Therefore, it needs to supplement a wheat germ cell-free extract with the fraction containing the limited initiation factors for the efficient protein translation, and this fortified cell-free system can be easily made by simple... 

Fig. 3. Autoradiograph of SDS-PAGE of in vitro translated dihydrofolate reductase (DHFR) in the wheat germ cell-free protein synthesis systems with (n) 4 pi of ribosome fiaction, (III) 4 pi of 0 -40 % ammonium sulfate fraction, or (IV) 4 pi of 40 - 60% ammonium sulfate fraction, respectively. Lane I is control dihydrofolate reductase produced in the normal wheat germ cell-free protein synthesis system.

Hawkins and Brash investigated some aspects of the biosynthesis of 11R-and 12R-HETE in eggs of S. purpuratus [192,194]. Based on experiments using octadeuterio-AA they showed that the 11- or 12-keto intermediate is not involved. Isolation and characterization of both HR- and 12R- hydro-peroxyeicosatetraenoic acids from incubations of desalted ammonium sulfate fractions of the egg homogenate with arachidonic acid support involvement of a lipoxygenase. 

In general, serum should be heat-inactivated by heating at 56°C for 15 min to inactivate complement components prior to ammonium sulfate fractionation. Ascitic fluid should first be filtered through a cushion of glass wool. 

Since ammonium sulfate fractionation is a crude procedure for antibody purification, this step may also be extended from 3 h to overnight for convenience. 

Since ammonium sulfate fractionation will also cause precipitation of other proteins, antibody concentrations obtained from absorbance measurements at 280 nm are only estimates. Alternatively, a sample of the dialyzed solution can be resolved on a SDS-polyacrylamide gel alongside a series of known concentrations of IgG. Staining the gel with Coomassie blue can then be used to estimate the amount of immunoglobulin obtained and can also give an estimate of purity. 

If the starting material is ascitic fluid or serum, then the sample should first be partially purified by ammonium sulfate fractionation followed by extensive dialysis or gel filtration (see Chapter 2). 

Ascites or serum should not be applied directly, but should be first purified by ammonium sulfate fractionation. 

Table III. Salicylic Acid Metabolite Production by a 50-65% Ammonium Sulfate Fraction Extracted from Oat Roots Exposed to...

Table 4.7. Ammonium sulfate fractionation of sera of different species...

Figure 3-4 Hypothetical behavior of a solution containing three proteins, A, B, and C, upon ammonium sulfate fractionation. The concentration of protein remaining in the solution is plotted against ammonium sulfate concentration (usually expressed as % saturation). Addition of ammonium sulfate to concentration c, will precipitate largely protein B, which can he removed by centrifugation. Addition of additional salt to c2 will precipitate largely protein C, while A remains in solution.

A cell extract was subjected to ammonium sulfate fractionation and the dialyzed protein was then poured through the affinity column which held the cytidine deaminase molecules because of their affinity for the cytidine structures that were bound to the agarose. 

Dialyze the serum (preferably ammonium sulfate fractionated) against 0 07M sodium phosphate buffer, pH 6 3, exhaustively (at least two changes over a 24 h period) at a ratio of at least 1 vol of sample to 100 vol of buffer. 

A large scale preparation of E. coli 045 was subjected to enzyme purification using the assay for 3,5-epimerase. Protamin sulfate precipitation, ammonium sulfate fractionation was followed by DEAE-chroma-tography. The fraction containing enzymatic activity, as measured by tritium exchange, was eluted from the DEAE column early. This fraction was incapable of producing any net synthesis of TDP-6-deoxy-L-... 

Selective adsorption on glass Ammonium sulfate fractionation Ammonium sulfate fractionation Ethanol precipitation Sonic disintegration Selective adsorption on cellulose phosphate CM-cellulose chromatography Acetone precipitation Ammonium sulfate fractionation Ethanol precipitation Selective adsorption on cellulose phosphate Ammonium sulfate fractionation Lysozyme and trypsin digestion Ammonium sulfate fractionation DEAE-cellulose chromatography Ammonium sulfate fractionation... 

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