Affinity techniques

Volume XXXIV. Affinity Techniques (Enzyme Purification Part B) Edited by William B. Jakoby and Meir Wilchek... 

Once the protein is modified to contain an alkynyl group at its C-terminal it can be used to covalently link to its click chemistry reactant partner, an azide on the surface of an array. Other azido molecules also can be conjugated with an alkyne-protein to facilitate the detection or capture of the protein using affinity techniques. For instance, an azido-fluorescein reagent can be used to detect fluorescently the expressed protein in complex samples or an azido-biotin... 

Schu(ller A, Bonfante P, Schnepf E, Mollenhauer D, Kluge M. Characterisation of the Geosiphon pyriforme symbiosome by affinity techniques confocal laser scanning (CLSM) and electron microscopy. Protoplasma 1996 190 53-67. 

Hermanson GT, Mallia AK, Smith PK (1992) Immobilised affinity techniques. Academic Press, New York... 

Jakoby, W. B. and M. Wilchek, eds., Affinity Techniques, Vol. XXXIV in Methods in Enzymotogy, Academic Press, New York, 1974. 

The type of ligand can be used to divide affinity techniques into various subcategories such as lectin, immunoaffinity, dye ligand etc. These techniques are placed as below [1]. 

In general, in affinity techniques Kd values are in the range of 10-8 to 10-4 M, but the Kd determined for free ligands can differ from those determined for immobilized ligands. Many authors report their data in terms of the association constant (Ka), which is equal to Kd 1-... 

Jakoby, W. B., Wilchek, M. Affinity techniques, in Methods in Enzymology, Vol. 34, Academic Press 1974... 

Knorre, D. G., Lavrik, O. I. Affinity labeling of aminoacyl-t-RNA synthetases, in Theory and Practice in Affinity Techniques, (ed. Sundaram, P. V., Eckstdn, F.) Academic Press, Lonson 1978, p. 169... 

In his pioneering contribution, Porath postulated that the histidine, cysteine, and tryptophan residues of a protein were most likely to form stable coordination bonds with chelated metal ions at near neutral pH [2]. To date, an analysis of several protein models [7] lend full to his original theory. Having said that, histidine, by far and away, plays the most prominent role in IMAC binding. In a very real sense, IMAC has subtly become synonymous as a histidine affinity technique. The absence of a histidine residue on a protein surface correlates with the lack of retention of that protein on any IDA-metal column. The presence of even a single histidine on a protein surface, available for coordination, results in retention of that protein on an IDA-Cu(II) column. Also, a protein needs to display at least two histidine residues on its surface to be retained on an IDA-Ni(II) column. Thus, beyond its role as a purification technique, IMAC has been used as a tool to probe the surface topography of proteins [6]. 

The application of biotinylated receptor substrates is another approach, incubating the labeled substrate with the receptors prior to isolation on an avidin-coated support. In such cases, biotinylation with a cleavable biotinylation reagent such as Sulfo-NHS-SS-biotin or NHS-Iminobiotin would be essential for recovery of the isolated receptor. Alternatively, the receptor could be recovered by substrate competition. Perhaps one of the major drawbacks to the application of affinity techniques is the relative low molecular weight or small size of the receptor substrates, making them difficult ligands to immobilize. However, affinity procedures have been applied to the purification of a number of different receptors although relatively little work has been reported on those involved in the processing of neurotransmittors, neuropeptides, and hormones [1,2]. 

Remove endogenous Apo-B by immuno-affinity technique (optionai)... 

Receptors Can Be Purified by Affinity Techniques or Expressed from Cloned Genes... 

Jacoby, W.B. and Wilchek, M. (eds.) (1974) Methods in Enzymology, Vol. 22, Enzyme Purification -Part B, Affinity Techniques, Academic Press, New York. 

Sundaram, P.V. and Eckstein, F. (eds.) (1978) Theory and Practice in Affinity Techniques, Proc. Int. Symp., Gottingen, 1978, Academic Press, London. 

Many other strategies involving affinity techniques have been devised. 

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