Affinity chromatography protein separation

Wu, H. and Bruley, D.F, Homologous Human Blood Protein Separation Using Immobilized Metal Affinity Chromatography Protein C Separation from Prothrombin with Application to the Separation of Factor IX and Prothrombin, Biotechnol. Prog., 15, 928, 1999. 

ATIII has been also used as a model protein to test a novel affinity chromatographic system capillary affinity chromatography [9]. Separation quality has been found equivalent to that observed with classical affinity chromatography, whereas the necessary protein amount is strongly reduced to the nanogram level. 

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. 

Wu H.P. and Bruley D.E. 1999. Homologous human blood protein separation using immobilized metal affinity chromatography protein C separation from prothrombin with apphcation to the separation of factor IX and prothrombin, Biotechnol. Prog., 15, 928-931. 

Affinity chromatography. A separation technique that exploits the high biological specificity and affinity of specific naturally occurring proteins for the analyte. 

Besides protein microarray proteome profiling, the term chemical proteomics (also chemoproteomics or pull-downs ) is mostly used in reference to the application of affinity chromatography protein purification when small molecules are the bait, and liquid chromatography separation of peptides precludes mass spectrometry as the universal readout (LC-MS/MS). 

Acyl-ACP thioesterase activity was assayed as described [1]. All purification steps were carried out at 4°C. Two hundred grams of frozen leek epidermis were ground to a powder in liquid nitrogen using a prechilled mortar and pestle. The protein was precipitated with 75% (w/v) ammonium sulfate, and then subjected to hydroxyapatite, Mono-S, Mono-Q, and ACP affinity chromatography. Proteins from each purification step were separated by SDS polyacrylamide gel electrophoresis according to Laemmli (1978)[3] and silver stained (Bio-Rad). 

The ELP expression system was compared to the conventional oligohistidme fusion, which is traditionally applied for purification by immobilized metal affinity chromatography (IMAC). Both techniques were shown to have a similar yield of the recombinant protein. The temperature-triggered approach offers a fast and inexpensive nonchromatographic separation with the possibility for larger scale purification. Although the ELP expression system may not be applicable to all types of recombinant proteins, numerous examples have already been shown [40]. 

The affinity chromatography on ConA - cellulose indicated the presence of small N-glycosylation of all forms of exopolygalacturonases present in carrot roots (unpublished results). This method was usefull for purification of these enzymes from other protein inpurities but was completely uneffective by separation of individual forms (Fig. 4). 

Protein affinity chromatography can be used for the separation of an individual compound, or a group of structurally similar compounds from crude-reaction mixtures, fermentation broths, or cell lysates by exploiting very specific and well-defined molecular interactions... 

Bound proteins are released by boiling for 2 min at 95° in the 4 x sample loading buffer and separated by SDS-PAGE for immunoblot analyses as described for Ni affinity chromatography in Section 2.4. 

Metal-chelate affinity chromatography is a powerful purification technique whereby proteins or other molecules can be separated based upon their ability to form coordination complexes with immobilized metal ions (Porath et al., 1975 Lonnerdal and Keen, 1982 Porath and Belew, 1983 Porath and Olin, 1983 Sulkowski, 1985 Kagedal, 1989). The metal ions are stabilized on a matrix through the use of chelating compounds which usually have multivalent points of interaction with the metal atoms. To form useful affinity supports, these metal ion complexes must have some free or weakly associated and exchangeable coordination sites. These exchangeable sites then can form complexes with coordination sites on proteins or other molecules. Substances that are able to interact with the immobilized metals will bind and be retained on... 

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