Chelation affinity binding

Antibiotics The AOAC has listed methods for sulfamethazine residues in swine tissues with determination either by GC-MS or GC-ECD of methylated derivatives and for sulfamethazine (and for the class of sulfonamides) in milk with determination by HPLC-UV. There is an AOAC method for the class of sulfonamide antimicrobials in animal tissues using solvent extraction and liquid partitioning with determination by TLC and fluorimetric scanning. For analysis of tetracyclines, AOAC describes methods based on buffer extraction from tissue samples and SPE (Cis) cleanup, or metal chelate affinity binding from milk samples, with determination in both cases by HPLC-UV. USDA/FSIS methods include (1) a method (similar to the AOAC GC-MS method for sulfamethazine) for confirmation of sulfonamide residues in edible tissues using solvent extraction and multiple liquid partitioning with determination of the methylated derivatives by GC-MS (2) methods for determination and confirmation of chloramphenicol in muscle by solvent extraction, liquid partitioning, and determination of the trimethylsilane (TMS) derivative by GC-ECD and GC-MS, respectively and (3) a method for determination of the beta-lactam antibiotic amoxicillin by aqueous extraction, cleanup by tricarboxylic acid precipitation, and ether extraction and formation of a fluorescent derivative for determination by LC. 

Kumar, A., Galaev, I. Y. and Mattiasson, B. (1998). Affinity predpitation of a-amylase inhibitor from wheat meal by metal chelate affinity binding using cu(n)-loaded copolymers of 1-vinylimidazole with N-isopropylacrylamide. Biotechnology and Bioengineering, 59, 695-704. 

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... 

Metal chelate affinity chromatography finds most prominent application in the affinity purification of recombinant proteins to which a histidine tag has been attached (described later). As protein binding occurs via the histidine residues, this technique is no more inherently useful for the purification of metalloproteins than for the purification of non-metalloproteins (a common misconception, given its name). 

In protein microarrays, capture molecules need to be immobilized in a functional state on a solid support. In principle, the format of the assay system does not limit the choice of appropriate surface chemistry. The same immobilization procedure can be applied for both planar and bead-based systems. Proteins can be immobilized on various surfaces (Fig. 1) (12). Two-dimensional polystyrene, polylysine, aminosilane, or aldehyde, epoxy- or thiol group-coated surfaces can be used to immobilize proteins via noncovalent or covalent attachment (13,14). Three-dimensional supports like nitrocellulose or hydrogel-coated surfaces enable the immobilization of the proteins in a network structure. Larger quantities of proteins can be immobilized and kept in a functional state. Affinity binding reagents such as protein A, G, and L can be used to immobilize antibodies (15), streptavidin is used for biotinylated proteins (16), chelate for His-tagged proteins (17, 18), anti-GST antibodies for GST fusion proteins (19), and oligonucleotides for cDNA or mRNA-protein hybrids (20). 

The stimulatory effect of flubendiamide on the Ca pump was also diminished in the calcium buffers comprised of calcium chelators with high and low calcium affinity [3]. The low affinity calcium chelator, diBr-BAPTA (Kd = 3.7 pM), evidently accelerated the catalytic cycles of Ca pump as in the case with A23187. The result also implies importance of luminal calcium, since the low affinity of this chelator could not interrupt the calcium association with high affinity binding sites (cytoplasmic site) on Ca -ATPase. In addition, the results also demonstrate... 

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