Subject affinity chromatography

FIGURE 15.3 Outline of experimental protocol used for ICAT differential protein expression profiling. Protein mixtures from two cell populations are labeled with light or heavy isotopic versions of a cleavable ICAT reagent. Labeled proteins are combined, subject to multidimensional separation by SCX, RP, and avidin affinity chromatography, then analyzed by tandem MS for peptide and protein identification. Based on the relative ratio of the two isotopically labeled peptides, a relative abundance of protein expression can be determined. 

MS can perform large-scale analyses of protein interactions. Interacting partners of proteins in complexes can be purified by several strategies including affinity chromatography and immunoprecipi-tation with antibodies specific to the bait protein. The purified components can then be subjected to LC-MS/MS and proteins within the complex identified. Using a variety of approaches including... 

In this chapter, we will survey the kinds of solid supports (substrates) and surface chemistries currently used in the creation of nucleic acid and protein microarrays. Which are the best supports and methods of attachment for nucleic acids or proteins Does it make sense to use the same attachment chemistry or substrate format for these biomolecules In order to begin to understand these kinds of questions, it is important to briefly review how such biomolecules were attached in the past to other solid supports such as affinity chromatography media, membranes, and enzyme-linked immxm-osorbent assay (ELISA) microtiter plates. However, the microarray substrate does not share certain unique properties and metrics with its predecessors. Principal among these are printing, spot morphology, and image analysis they are the subjects of subsequent chapters. 

The aqueous or organic extract obtained at this step of analysis may be a very dilute solution of the analyte(s) of interest. It may also contain coextractives, which, if allowed in the final extract, will increase the background noise of the detector, making it impossible to determine trace level concentrations of the analyte(s). To reduce interferences and concentrate the analyte(s), the primary sample extracts are subjected to some kind of cleanup including liquid-liquid partitioning, solid-phase extraction, matrix solid-phase dispersion, online trace enrichment, affinity chromatography, immunoaffinity chromatography, and ultrafiltration. In many instances, more than one of these procedures may be used in combination to increase extract purification. 

Membrane fractions of several cell lines derived from immunocompetent cells were previously analyzed by photoaffinity labeling (Williamson et al., 1995) and the key enzyme in pyrimidine biosynthesis, DHODH, was identified as one of the major targets of Leflimomide. For further elucidation of its mode of action, the cytosolic fraction of the monocyte/macrophage cell line RAW 264.7 was subjected to affinity chromatography to identify all cytosolic binding partners of Leflunomide. The cytosolic preparation was applied to an affinity column where the Leflunomide derivative A 95 0277 was coupled as a ligand (Figure 10.2). 

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