Affinity chromatography variations

The mechanisms described above form the basis for the chromatographic modes described in Chapter 2, namely, normal-phase, reversed-phase, size-exclusion, ion-exchange, and affinity chromatographies. However, other modes that are variations of those mentioned above, such as hydrophobic-interaction chromatography (HIC), chiral, ion-exclusion, and ion-pair chromatographies are also used and will be mentioned. 

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. 

Antibody Characterization. Typical c-ELISA s for LLNL-Hept-2 using heptachlor as competitor (IS samples run over a 6-month interval) are presented in Figure 2. These data indicate that the average Iso tor heptachlor occured when 3.0 ng of analyte was added to the reaction. A standard deviation of 7% was observed causing the Iso values to range between 1.9 and 4.S ng/well. Similar variations were observed with other competitors. The LLNL-Hept-2 antibody used in the above experiments, and all subsequent experiments, was purified from ascites fluid by affinity chromatography on Sepharose Protein G (see methods section). 

Recent advances in the purification of DNA-binding proteins have been due primarily to the use of the specific sequence of the binding site as a ligand for affinity chromatography. Different variations of this basic principle have been developed in several laboratories, and all have been found to function effectively. 

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