A multi-dimensional chromatographic approach was developed to measure the free fractions of drug enantiomers in samples that also contained a binding protein or serum. This method, which combined ultrafast affinity extraction with a chiral stationary phase, was demonstrated using the drug warfarin and the protein human serum albumin.

Human transferrin receptor is a disulfide-linked homodimer of 90-kDa glycoprotein subunits, capable of binding two transferrins. We report a new high yield affinity purification protocol for transferrin receptor from placenta which produces 3-4 mg of highly purified protein. Trypsin cleaves the protein at arginine-121, producing a stable fragment that contains 95% of the extracytoplasmic sequen...

Affinity chromatography using agarose-bound lectins was used to isolate erythropoietin from crude preparations of sheep plasma and human urinary erythropoietin. On the basis of previous estimates of the sugar content of the hormone, six lectins (wheat germ agglutinin, phytohemagglutinin, Ricinus communis 120, soybean agglutinin, concanavalin A, and limulin) were chosen for study. Only wheat ger...

We describe a fast and simple one-step affinity-purification method for the isolation of specific RNA-binding proteins. An in vitro-transcribed hybrid RNA consisting of an aptamer sequence with high binding specificity to the antibiotic streptomycin and a putative protein-binding RNA sequence is incubated with crude extract. After complex formation, the sample is applied to an affinity column c...

The affinity chromatographic separation procedure was tested for its utility in quantitating the affinity of proteins to both insolubilized ligand and corresponding competing soluble ligand. An expression has been derived that (1) allows the determination of binding constants for the interaction of a soluble protein species with an affinity chromatography matrix involving active site binding, (...

The peak decay method is an affinity chromatographic technique that has been used to examine the dissociation of solutes from immobilized ligands in the presence of excess displacing agent. However, it can be difficult to find a displacing agent that does not interfere with detection of the eluting analyte. In this study, a noncompetitive peak decay method was developed in which no displacing a...

The efficacy of activation methods and coupling were studied in the context of performance in batch and fixed bed binding experiments utilizing cell culture fluids or blood plasma as feedstock. Conclusions were drawn regarding selection of solid phase according to pore size, rigidity, pH stability, Chemistry of derivation and activation, and gross concentration of immobilized ligand require...

Peak profiling and high-performance columns containing immobilized human serum albumin (HSA) were used to study the interaction kinetics of chiral solutes with this protein. This approach was tested using the phenytoin metabolites 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) as model analytes. HSA columns provided some resolution of the enant...

Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monocl...

The expression of cell-surface haemopexin (Hx) receptors on human cytotrophoblasts was assessed by using four different Hx species purified from plasma: human Hx isolated by wheatgerm-affinity chromatography, human Hx isolated by haem-agarose-affinity chromatography and rabbit and rat Hx, also isolated by haem-agarose-affinity chromatography. About 3500-7000 high-affinity (Kd 0.34-0.85 nM) rece...