Affinity chromatography procedure

Four rabbits were immunized each with 0.3 mL of the nonviable suspension of cells administered daily intravenously for 3 days, followed by a rest period of 4 days. This schedule was repeated for 5 weeks. After a 1-week rest period, a second administration of vaccine was used following the foregoing schedule. Blood samples were drawn weekly and serum was obtained. In 6 weeks the sera of the rabbits showed a high titer of antibodies directed at the cell-wall polysaccharide. The antisera from each rabbit were maintained separately. The antibodies were isolated from the serum by an affinity-chromatography procedure employing adsorbtion on lactosyl-... 

Torpedo AcChR was purified by an affinity chromatography procedure [II] DEAE-cellulose chromatography of the purified material, followed by concentration on a PM 10 dia-flow membrane yielded a product with a specific activity of 10 nmoles aBgt binding sites per mg protein (or 1 binding site per 100000 daltons of protein). 

Bacterial luciferase can be purified by various conventional chromatographic techniques (6) and by using a series of HPLC columns (7). One of the new purification techniques relies on an affinity matrix composed of a luciferase inhibitor, 2,2-diphenylpropylamine, conjugated to Sepharose (13,35). The affinity chromatography procedure is reported to give a routine yield (60%) of 600—700 mg of over 90% pure luciferase from a 500 g batch of cell paste. 

Crude chloroform-methanol-water (30 60 8, v/v) extracts of immunostainedTLC bands were analyzed without further purification by nanoelectrospray low-energy mass spectrometry. The authors showed that this effective PLC/MS-joined procedure offers a wide range of applications for any carbohydrate-binding agents such as bacterial toxins, plant lectins, and others. Phenyl-boronic acid (PBA) immobilized on stationary support phases can be put to similar applications. This technology, named boronate affinity chromatography (BAC), consists of a chemical reaction of 1,2- and 1,3-diols with the bonded-phase PBA to form a stable... 

From the foregoing discussion, it is apparent that heparin may be subdivided into an unlimited number of fractions when different separation approaches are applied to each fraction obtained by another procedure. More than a hundred fractions have been obtained by sequential use of affinity chromatography on antithrombin, precipitation with barium, and isoelectrofocusing.214 Although these fractions can scarcely be referred to as species, such an extensive fractionation stresses the concept of the heterogeneity of heparin, and the influence of minor differences in chemical constituents, or chain length, or both, on the physicochemical (and, conceivably, biological) properties of this polysaccharide. 

This method was used, for example, for the solid-phase immunoassay of thyroxine (affinity chromatography). Various activation methods (CDI, periodate, and cyanogen bromide procedures) were compared with each other for coupling antibodies to magnetizable cellulose/iron oxide solid-phase particles. 211]... 

Figure 6.2 A Likely purification procedure for tPA produced in recombinant E. coLi cells. The heterologous product accumulates intracellularly in the form of inclusion bodies. In this particular procedure, an ultrafiltra-tion step is introduced on several occasions to concentrate the product stream, particularly prior to application to chromatographic columns. Lysine affinity chromatography (Lys-chromatography) is employed, as tPA is known to bind immobilized Lysine molecules. Adapted with permission from Bio/Technology (1993), 11, 351... 

It is not normally prudent to employ biospecific affinity chromatography as an initial purification step, as various enzymatic activities present in the crude fractions may modify or degrade the expensive affinity gels. However, it should be utilized as early as possible in the purification procedure in order to accrue the full benefit afforded by its high specificity. 

Recently, Noort et al developed a procedure that is based on straightforward isolation of adducted BuChE from plasma by means of affinity chromatography with a procainamide column, followed by pepsin digestion and LC/electrospray tandem MS analysis of a specific nonapeptide containing the phosphonylated active site serine-198 residue (5). This method surpasses the limitations of the fluoride-reactivation method, since it can also deal with dealkylated ( aged ) phosphonylated BuChE. The method allowed the positive analysis of several serum samples of Japanese victims of the terrorist attack in the Tokyo subway in 1995. Furthermore, the method could be applied for detection of ChE modifications induced by, e.g., diethyl paraoxon and pyridostigmine bromide, illustrating the broad scope of this approach. This new approach... 

P-Mannanases are generally prepared by conventional chromatographic procedures, which can lead to high yields of enzyme, but in some cases only a poor state of purity. Small quantities of highly purified p-mannanases have been prepared by substrate affinity chromatography of partially purified enzymes on a column of glucomannan immobilised on aminohexane Sepharose 4B (6). The action patterns of enzymes described in this paper were determined using enzymes purified by this latter procedure. 

Furthmayr, H. (1982). Immunization procedures, isolation by affinity chromatography and seriological and immunochemical characterization of collagen-specific antibodies. In Furthmayr, H., ed. immunochemistry of the Extracellular Matrix, Vol. 1. CRC Press, Boca Raton, FL. 

Specialized Purification Procedures Purification of membrane proteins, 182, 499 purification of integral membrane proteins, 104, 329 reconstitution of membrane proteins, 104, 340 purification of DNA-binding proteins by site-specific DNA affinity chromatography, 182, 521 purification of glycoproteins, 182, 529 purification of multienzyme complexes, 182, 539. 

A3, H2, S16) In general, immunological procedures have been employed for assessment of the purity of the various lipoprotein preparations. At this time, however, the availability of specific antisera may permit the application of immunological procedures in preparative work, particularly by the technique of affinity chromatography. 

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