Protein affinity chromatography Subject

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. 

In order to measure susceptibility to oxidation without the need to isolate the lipoproteins, methods have been developed for oxidizing whole serum or plasma and measuring diene formation (R2, S4). Such approaches may be subject to error as a result of variation in other oxidizable plasma components such as bilirubin, albumin, fibrinogen, and uric acid. A method that uses heparin affinity chromatography to separate LDL and intermediate density lipoproteins (IDL) from other serum proteins was described by Vinson et al. (V3, V5) and was later better standardized (K3). This approach has been shown to reflect susceptibility to oxidation in animal and human plasma under a variety of conditions (K3, V5). The heparin separation procedure is detailed in the following text. 

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