Affinity chromatography precipitation with ammonium sulfate

Initially, the antibodies should be purified prior to prepare the immunoaffinity column. Precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration chraoma-tography or affinity chromatography may be employed with the aim of antibody purification. Activated beads which are coated with bacterial proteins A or G may be used as the support material. Some parameters may be changed for the elution of the sample solution for example the ionic conditions of mobile phase may be changed or chaotropic buffers may be used [11]. 

P.p. are usually isolated on a preparative scale without loss of biological activity, by precipitation with ammonium sulfate, ethanol or rivanol. To an increasing extent, however, these methods are being replaced, even on an industrial scale, by efficient column methods, such as gel filtration, ion exchange, affinity and immunoadrarption chromatography, and by ultrafiltration. 

Antibody sera obtained from immunized animals can in some cases be directly used for further experiments. Further purification and enrichmenL however, are often necessary to increase the specificity and overall affinity of fhe purified antibodies. Simple enrichment of fhe antibodies can be achieved by precipitation wifh ammonium sulfate [55] or by chromatography wifh protein A [56] or protein G, bacterial ceU waU proteins fhat specificaUy bind to fhe Fc portion of immunoglobulins. A more specific enrichment is achieved by affinity chromatography with an antigen column. 

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.

This conception works out as shown in Scheme 10 CTP 23 formed by the above described sequence is directly consumed by -acetyl neuraminic acid 26 under the catalytic influence of cytidine-5 -monophosphosialate synthase (E.C. 2.7.7.43). This enzyme is isolated from calf brain by ammonium sulfate precipitation (2 5) and subsequent affinity chromatography. The stationary phase consists of CNBr-activated Sepharose 4B reacted with p-[3-(2-amino ethylthio)propyl]-iV-acetyl neuraminic acid 27, which is synthesized by radiating a mixture of the allyl glycoside and cysteamine to achieve radical C-S bond formation (24), The behavior of methyl p-N-acetyl-neuraminic acid as an inhibitor is in accordance with Zbiral s findings (25), where the methyl a-glycoside has been shown to compete with the native substrate for the enzyme, and thus 27 is recommended to be an ideally suited ligand (Scheme 9). A typical analytical run is shown in Scheme 9. Due to elution of the protein fraction by a salt gradient, the transfer to a preparative scale is rather difEcult denaturation occurs and thus a drop in activity down to 6% is observed. 

RNR activity is readily detectable in soluble cell-free extracts of S. shibatae, S. solfataricus, and P. furiosus, incubated with cytidine diphosphate (CDP), dithio-threitol (DTT), and adenosylcobalamin (AdoCbl), at high temperatures (70°-80°). Sodium acetate stimulates the activity and is included in the assay. Acetate probably acts as an allosteric effector. The activity is not air-sensitive and purification can be achieved aerobically at room temperature, reflecting the extreme stability of the enzyme. The activity can be purified 3000-fold in three steps (ammonium sulfate precipitation, phenyl-Sepharose and dATP-Sepharose chromatography) (Table I). The key purification step is affinity chromatography on dATP-Sepharose (400-fold purification), a method used successfully for the purification of several other RNRs.3i.32 Binding to this ligand is an indication that the thermophilic RNR is... 

Z to (diH)Z have been conducted (Plant Physiol, submitted). The reaction is NADPH-dependent and does not require the presence of ATP and cations (Table 2). The enzyme has been partially purified using ammonium sulfate precipitation, anion exchange and affinity column chromatography. The affinity of the enzyme for i Ade (N -(A -isopentenyl)adenine), the unhydroxylated counterpart of Z, is negligible. Preliminary estimates of Z reductase activities varied substantially between species, with high activity in P. coccineus and P. vulgaris embryos but only marginally detectable activity in P. lunatus. 

Anti-PNP antibodies are produced by rabbits immunized with PNP20-bovine y-globulin and are purified from a 50% ammonium sulfate precipitate of serum by affinity chromatography on t-2,4 dinitrophenyllysine agarose with elution by 2,4-dinitrophenylglycine/ The preparation is dialyzed against 0.2 M sodium borate-0.15 M NaCl, pH 8.0, to remove hapten. 

Acyl-ACP thioesterase activity was assayed as described [1]. All purification steps were carried out at 4°C. Two hundred grams of frozen leek epidermis were ground to a powder in liquid nitrogen using a prechilled mortar and pestle. The protein was precipitated with 75% (w/v) ammonium sulfate, and then subjected to hydroxyapatite, Mono-S, Mono-Q, and ACP affinity chromatography. Proteins from each purification step were separated by SDS polyacrylamide gel electrophoresis according to Laemmli (1978)[3] and silver stained (Bio-Rad). 

Extraction of spinach NADP-MDH was achieved in a Waring blendor by 50 mM tris-HCl buffer, pH 7.5, 2 C. After centrifugation, the crude extract was acidified to pH 4.8 with formic acid. The supernatant after centrifugation, was precipitated with 55 % ammonium sulfate. The pellet was dialyzed against 5 mM phosphate buffer (pH 7) and submitted to DEAE-cellulose chromatography (Fig. 1 a). Peak one is MDH A with a fraction of MDH B. The purification of MDH A is carried out by an affinity chromatography on matrex gel and on 2 ,5 -ADP-Sepharose. Peak two of DEAE-cellulose is MDH B,... 

---