Affinity chromatography loading procedures

Figure 3. Selection of ECP subpopulations forjprogressive iterations of the cascade procedure by silver stained SDS-PAGE. Lane 2 in each panel shows the entire ECP mixture used as the column load and lane 3 shows the column flowthrough fraction used for the next injection. Panel A demonstrates the affinity chromatography performed with day 14 antisera, Panel B with day 28 antisera and Panel C with day 42 antisera. The arrow shows ECPs depleted by the early antibodies. The progression of the immune response is clearly apparent although it is clear not all of these proteins are equally immunogenic. A 50 Kd protein has saturated its respective antibody and begun to flow through the column (Panel C, lane 4). Reproduced with permission from Ref. 24. Copyright 1989 The Humana Press Inc.

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. 

Speciation of Cr as part of biomolecules consists in applying well known biochemical procedures, such as gel permeation chromatography, anion - cation chromatography, affinity chromatography, capillary electrophoresis, ultrafiltration, to be followed on the one hand by the determination of the exact amount of a specific protein and on the other hand by the quantitative determination of the chromium present in the fractions (Cornells, 1990). This is not an easy task. All the commercially available buffers are unfortunately loaded with Cr. The separation medium (e.g. the gel) may be another possible source of error. The contaminating Cr atoms are liable to form fortuitous bindings with the proteins which possess many free ligands. 

The most conventionally used macroscale affinity-based cell separation system is cell affinity chromatography (CAC). These systems can provide relatively high purity (>95 %) and high throughput (10-10 cells/h). CAC procedures can be divided into three consecutive steps loading,... 

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