Affinity chromatography immobilization procedures

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... 

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... 

The preselected RNA pool is either allowed to flow through a column with immobilized target (affinity chromatography) or is incubated with the matrix carrying the target (batch procedure). 

Table 1. Purification of EcaL-ASNasc using a two-step procedure employing cation-exchange chromatography on CM-Sepharose and affinity chromatography on immobilized L-Asn. The procedure was carried out at 4°C...

A chromatographic fractionation procedure that uses a matrix consisting of a metal ion tightly bound to a matrix. Nickel is the most common metal ion used but there is use of copper and other transition metals. See Porath, J. and Ohn, B., Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities... 

Transhydrogenase was also purified somewhat later and independently by a more complicated procedure which, however, also produced the 115000 molecular weight polypeptide [102]. Subsequently, additional improved methods have been published, which involve the use of immobilized antibodies [103] and affinity chromatography on immobilized NAD" [104] or NADP" [105]. With all preparations reconstituted transhydrogenase was shown to be a proton pump by both an indirect assay using 9-aminoacridine as pH probe for the interior space of the vesicles [106], and a direct assay using a pH electrode [107]. 

Desalting can be achieved by precipitation with ethanolic atmnonium acetate [4], solid-phase extraction (SPE), microdialysis [13], Fe " -loaded immobilized metal affinity chromatography (IMAC) [14], cation-exchange coluttms (SCX) [15-16], or combinations thereof [17-18]. The SCX procedure can be applied on-hne with direct infusion of the effluent into ESl-MS [15-16]. The mobile phase applied is similar to the one applied in LC-MS, i.e., 50% acetonitrile in 10 mmol/1 aqueous TEA. 

Protease-free preparation of CNTs can be obtained using an immobilized-metal-ion affinity chromatography (IMAC) step (Ros-setto et a/., 1992). This procedure is also useful for the purification of He, the 50 kDa carboxy-terminal part of the heavy chain of TeTx, which shows an identical retention time. However, IMAC-chromatography cannot be used for the purification of BoNT/D because this serotype is not retained. To obtain a protease-free BoNT/D preparation, an ion-exchange chromatography procedure was used (Schiavo and Montecucco, 1995). After freezing in liquid nitrogen, purified CNTs are stored at -80°C. 

Estevez and Prestwich converted the diol 249(R=MPM), obtained according to the same sequence, into a photoaffinity probe 251 with an azido function for Ins(1,3,4,5)P4 (Scheme 4-18). 9 2 The aminopropyl ester obtained after the hydrogenolysis procedure was also immobilized to a resin for affinity chromatography. 

---