Steroid affinity chromatography

Partially purified receptor preparations have been used for production of polyclonal and monoclonal antibodies to receptors. If these antibodies are available they might be used for immunoaffinity chromatography of receptors. This procedure permits rapid isolation of highly purified receptors and has been used, e.g., for separation of different phosphorylated forms of progestin receptors [30], The right choice of a suitable reagent for elution of receptors from the immunoaffinity column in a form still able to bind the steroid was crucial in these studies. 

Classical characterization procedures of non-purified receptors were based on behavior of the radioactive label of the steroid bound to the receptor. Sucrose gradient centrifugation and gel filtration have been used for estimation of size and molecular mass (Table I, Section 2.2). Iso-electric focussing under non-denaturing conditions revealed iso-electric points between 4.5 and 6.0 for the monomeric forms. 

Baulieu, E.-E, Alberga, A., Jung, I., Lebeau, M.-C., Mercier-Bodard, C., Milgrom, E., Raynaud, 

Raynaud-Jammet, C., Rochefort, H., Truong, H. and Robel, P. (1971) Recent Progr. Horm. Res. 27, 351-412. 

(1976) In Receptors and Mechanism of Action of Steroid Hormones, Part I, Ch. 1 (Pas-qualini, J.R., ed.) pp. 1-40. Marcel Dekker, New York. 

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Vol. XXXrV [1], The Literature on Affinity Chromatography. M. Wilchek and W. B. Jakoby. Vol. XXXVI [1]. Theory of Protein-Ligand Interaction. D. Robard and H. A. Feldman. Vol. XXXVI [2]. Use of Specific Antibodies for Quantification of Steroid Hormones, G. D. Niswender, A. M. Akbar, and T. M. Nett. 

Affinity chromatography can be applied as a purification technique to any pair of biologically complementary substances, for example, enzymes and inhibitors, steroids and binding proteins, antigens and antibodies. Insolubilization of one member of the pair yields an affinity column specific for the second member of the pair. Affinity chromatography can also be used to concentrate dilute solutions of purified substances and removal of soluble substances which are present as contaminants in low concentration. 

Various types of column can be combined. Sequential analysis with liquid chromatography can first pass the sample through a gel to separate constituents according to their molecular size, followed by some form of affinity chromatography using partition or adsorption. A two-column GLC system for steroid analysis has been described (H22), in which the sample is first introduced to a high-capacity low-resolution column for a preliminary separation. After a suitable time, the flow of gas is re-... 

Steroid hormones are often present in cells in low concentrations. This makes them difficult to isolate and identify. It is sometimes easier to isolate the proteins to which they bind by using affinity chromatography. (Refer to Biochemical Methods 5.1.) Explain how you would use this technique to isolate a protein suspected of steroid hormone binding. 

If you work with solutes such a steroids, isomers or naphthalene derivatives, or in a separation mode such as ion exchange, affinity chromatography, chiral chromatography etc., you should certainly check the influence of the temperature on the selectivity, but not just in the range 30 0 °C. 

As early as 1951, affinity chromatography was used for the separation of anti-hapten antibodies (Campbell et aL, 1951). Shortly afterwards, Lerman (1953) developed a similar method for purification of tyrosinase. The recent applications of affinity chromatography are too numerous to be detailed here, but broadly speaking, the method has been used for separation/purification of proteins, sugars and their derivatives, nucleic acids, nucleotides and their derivatives, amino acids and peptides, and various other systems that include thiols and disulfides (ligands-thiols or disulfides or organomercurial supports), steroidal hormones, coenzymes, vitamins, morphine and related drugs, antibiotics, protein receptors, and antibodies. 

G. Giraudi, C. Baggiani, Strategy for fractionating high-affinity antibodies to steroid hormones by affinity chromatography, Analyst 121 (1996) 939-944. 

Purification of the steroid-active isoenzyme of alcohol dehydrogenase by affinity chromatography Isolation of ribonucleotide reductase by affinity chromatography Investigations of the effects of different spacer arms on the affinity chromatography of alkaline phosphatase Purification of P-450 haemeproteins of Rhizobium japonicum by affinity chromatography... 

The preferred tracer in commercially available RIA kits for labeled testosterone is I. The size of the iodinated group and the site of linkage may greatly affect assay affinity and specificity. Some methods use solvent extraction or chromatography, or both, to remove interferences from other steroids and some drugs. These prefiminaiy steps also permit concentration of the sample when low concentrations of testosterone are expected. Like automated assays, manual RIA assays perform better at higher concentrations, with questionable utility in female and prepubertal subjects. ... 

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