Ammonium sulfate precipitation with

The gaseous ammonia is passed through electrostatic precipitators for particulate removal and mixed with the cooled gas stream. The combined stream flows to the ammonia absorber where the ammonia is recovered by reaction with a dilute solution of sulfuric acid to form ammonium sulfate. Ammonium sulfate precipitates as small crystals after the solution becomes saturated and is withdrawn as a slurry. The slurry is further processed in centrifuge faciHties for recovery. Crystal size can be increased by employing one of two processes (99), either low differential controUed crystallization or mechanical size enlargement by continuous compacting and granulation. 

Ammonium Sulfate Precipitation. The extract was made up to 40% saturation with the slow addition, with stirring, of ammonium sulfate at 4°C. After several hours, the precipitate was removed by centrifugation at 30,000 g for 30 min and the supernatant retained. It was brought to 100% saturation in similar conditions, the precipitate was collected by centrifugation, dissolved in the minimum of distilled water, dialyzed against water and then against 1% glycine, and lyophilized. 

Figure 2. Gel filtration. The dry residue obtained after ammonium sulfate precipitation was redissolved in 50 mM phosphate buffer, pH 7.4 0.15 M NaCl 0.013 % sodium azide, which was loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fractions were collected. The fractions were assayed for protein content (— ) and PNL activity (- - ).

Figure 5 shows the pattern of lyase isoenzymes along the purification process at first, three bands with lyase activity (pis 9.20, 9.00 and 8.65) were detected in the ammonium sulfate precipitate (B 1) in the peak eluted from the Superdex 75HR1030 column, only one band with lyase activity was detected, that correspond to the PNL with pi 9.20 (B 2), but more proteins were detected by silver staining (A 2). 

The development of modem separation techniques has affected the purification procedures employed for D-galacturonanases. Fractional precipitation with ammonium sulfate and with organic solvents are now used only in combination with new separation techniques. To separate fractions having D-galacturonanase activity, adsorption to pectate or calcium pectate gel has been used in several instances.157-207... 

Jayle, Herman-Boussier, and Moretti have shown that different types of human Hp in amounts large enough for analysis can be prepared by fractional ammonium sulfate precipitation combined with a rather conventional, preparative electrophoresis in an acetate buffer of pH 5.8. Schultze and Heide (S2) utilize a more complicated procedure, in which preparative electrophoresis (acetate buffer, pH 4.4) is likewise the final step. 

For most reactions, one can use 1-2 mM acetyl phosphate and 1-2 international units of acetate kinase. The enzyme is usually supplied as a crystalhne suspension in 3 M ammonium sulfate, and 10 mM ammonium ion is inhibitory. Therefore, a useful practice is to snip off 0.5 cm from a disposable Eppendorf micropipette tip to facih-tate removal of 10-20 microliters of the crystalline suspension then spin down the enzyme in a 1.5 ml disposable conical plastic centrifuge tube, and remove the ammonium sulfate solution with a wick of twisted Kim-wipe. The enzyme precipitate can now be taken up directly into your working buffer. Note Acetate kinase is inactivated by cold exposure, but incubation with 10 M ATP or GTP reactivates the enzyme if warmed to room temperature for 5-10 min. 

Specific Antibody Determination. Serum samples were prepared from each bleed by centrifugation to remove clotted material. 100 ul of the sera was incubated for 30 minutes with sufficient H-STXOL to provide a ca. 20 fold excess of hapten to the anticipated quantity of specific binding sites. The radioactivity of the protein pellet was determined after ammonium sulfate precipitation. After correction for a small amount of non-specific adsorption of label by control sera proteins the mg/ml of specific antibody in the sample was calculated. 

Figure 6. Affinity chromatography of EGD from Clostridium thermocellum. Nucleic acid preparation, heat treatment and ammonium sulfate precipitation (0-70%, 70-100%) were carried out as described (10). The final precipitate ( 50 mg protein), dissolved in 50 mM sodium acetate, pH 5.0, was applied (after centrifugation) on the affinity column (2 x 25 cm) (4 -aminobenzyl l-thio-/ -cellobioside coupled to Sepharose 4B) (11). Protein was monitored at 280 nm and the activity of the fractions (2 ml) determined using 2 -chloro-4 -nitrophenyl / -cellobioside (pH 6.5, 25°C) as described in the text. Elution with 10 mM G2 was started as indicated.

A number of approaches have been used to determine the amount of HA protein in a sample. The most successful of these is based on adding a fixed amount of TA to a sample (Thompson and Forward, 1969) after incubation, the turbidity is measured and the increase in turbidity observed is presumed to be proportional to the amount of HA protein in the sample. This method has the advantage that only substances able to form haze with polyphenols respond. The saturated ammonium sulfate precipitation limit (SAPL) method has also been widely used, but is far inferior in providing useful information (Berg et al., 2007 Siebert et al., 2005). 

In vitro studies were conducted with enzymes extracted from peanut (7), pea (. ), and onion (9). The enzymes were fractionated by ammonium sulfate precipitation, dialyzed, and stored frozen until used. The enzymes were assayed for various activities as described In the Results and Discussion. 

Figure B3.1.1 A 15% SDS-polyacrylamide gel stained with Coomassie brilliant blue. Protein samples were assayed for the purification of a proteinase, cathepsin L, from fish muscle according to the method of Seymour et al. (1994). Lane 1, purified cathepsin L after butyl-Sepharose chromatography. Lane 2, cathepsin L complex with a cystatin-like proteinase inhibitor after butyl-Sepharose chromatography. Lane 3, sarcoplasmic fish muscle extract after heat treatment and ammonium sulfate precipitation. Lane 4, sarcoplasmic fish muscle extract. Lanes M, low-molecular-weight standards aprotinin (Mr 6,500), a-lactalbumin (Mr 14,200), trypsin inhibitor (Mr 20,000), trypsinogen (Mr 24,000), carbonic anhydrase (Mr 29,000), gylceraldehyde-3-phosphate dehydrogenase (Mr 36,000), ovalbumin (Mr 45,000), and albumin (Mr 66,000) in order shown from bottom of gel. Lane 1 contains 4 pg protein lanes 2 to 4 each contain 7 pg protein.

In view of the high stability of the enzyme most samples have been prepared by the procedure described by Kunitz (16) and modified by McDonald (17) to remove all traces of proteolytic activity. During this procedure the minced bovine pancreas is exposed to 0.25 N sulfuric acid, ammonium sulfate precipitation, 10 min at 95°-100° and pH 3, and, finally, reprecipitation. The product can be crystallized it was also shown later to contain a number of components all with ribonuclease activity. A practical summary of all details is given by Kunitz and McDonald (18). 

The concentration procedure in Step (5) was developed by trial and error. Other methods tried were solvent precipitation with acetone and/ or alcohol, Amicon ultrafiltration with a PM-10 membrane, lyophilization alone, and ammonium sulfate precipitation alone. 

After homogenization, it is often advantageous to perform a salt precipitation, most commonly with ammonium sulfate. The purpose of this precipitation is the separation of cell debris and nucleic acids rather than the purification of the target protein from impurities. Whereas the purification factor of ammonium sulfate precipitation is usually around only 1.5 to 2, the separation of non-proteineous impurities and stabilization of the target protein in ammonium sulfate usually provide sufficient benefit to include this step in any purification protocol. 

ALS was isolated from barley seedlings as a 0-33% Ammonium Sulfate precipitate and examined for inhibition by TP. It is apparent from Figure 5 that the enzyme is very sensitive to the compound. The 1(50) value (concentration required for 50% inhibition) was calculated to be 0.047 uM. This value is within the range reported for CS tested against ALS from different species (19). Imidazolinones are less potent with 1(50) values in the range 2-12 uM (26). ALS isolated from several species and their 1(50) values for TP is shown in Table I. 

The overall recovery of the enzyme was >60 % with a 235-fold purification. The final preparation, however, was not homogenous. Barley has two isoforms of ALS which can be separated on a phenyl agarose column immediately after the ammonium sulfate precipitation. One of the forms does not bind to the column and was too unstable to attempt purification. The details of purification in Table III pertain to the fraction with affinity for phenyl agarose. 

Purification of Monoclonal Antibody. Immunoglobulins were precipitated from the pooled ascites by addition of an equal volume of saturated ammonium sulfate [50% (NH4)2S04]. The precipitate was collected by centrifugation (20 min 10,240 X g), dissolved in 0.01 M sodium phosphate (pH 6.8), and reprecipitated. After the second ammonium sulfate precipitation, the pellet was dissolved in a minimum volume of 0.01 M sodium phosphate (pH 6.8) and centrifuged for 10 min at 10,600 X g). The resulting supemate was applied to a P6G, gel filtration polyacrylamid, column (Bio-gel Biorad, Rockville Center, NY 1.5 X 40 cm). Fractions containing protein were pooled and applied to a hydroxyapatite column that had been equilibrated with 0.01 M sodium phosphate (pH 6.8). Proteins were eluted with a linear gradient of 0.01 to 0.3 M sodium phosphate. 

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