Affinity chromatograph

Affinity chromatographic procedures have been widely accepted for purification of biological macromolecules48). This technique is also one of the most convenient... 

Bog-Hansen, T.C., Prahl, P., and Lowenstein, H. (1978) A set of analytical electrophoresis experiments to predict the results of affinity chromatographic separations. Fractionation of allergens from cow s hair and dander./. Immunol. Meth. 22, 293. 

Enzymes. The specificity of an enzyme for its substrate, coenzyme or competitive inhibitor provides the basis for many affinity chromatographic separations. Enzymes may be extracted and purified using insolubilized substrates, coenzyme or inhibitors. Less frequently, enzymes are used as the ligands. 

Immobilized enzymes may be used in affinity chromatographic methods but their use as catalysts may be in either the production or removal of compounds in chemical processes or as analytical tools. Many substrate assays can be performed using enzymes immobilized on a variety of surfaces, e.g. glass beads, plastic or nylon tubing. Alternatively they may be incorporated into gel or microparticulate layers on dry strips or slides. 

Most eukaryotic mRNA molecules have up to 250 adenine bases at their 3 end. These poly (A) tails can be used in the affinity chromatographic purification of mRNA from a total cellular RNA extract. Under high salt conditions, poly (A) will hybridize to oligo-dT-cellulose or poly(U)-sepharose. These materials are polymers of 10 to 20 deoxythymidine or uridine nucleotides covalently bound to a carbohydrate support. They bind mRNA containing poly (A) tails as short as 20 residues. rRNA and tRNA do not possess poly (A) sequences and will not bind. After washing the mRNA can be eluted with a low salt buffer. 

O. Miyawaki and T. Yano, Dynamic affinity between dissociable coenzyme and immobilized enzyme in an affinity chromatographic reactor with single enzyme, Biotechnol. Bioeng., 39, 314-319 (1992). 

A second method of incorporating phase into the selection process is to immobilize the target. Essentially an affinity chromatographic method, this allows nonbound library constituents to be washed away, leaving the selected compound(s) bound to resin. Eliseev s guanidine resin and Still s peptide-bearing beads, both discussed above in the context of exchange reactions, are examples of these. 

An Affinity Chromatographic Method for the Purification of Some Cellulolytic Enzymes... 

Q Yang, X-Y Liu, M Hara, P Lundahl, J Miyake. Quantitative affinity chromatographic studies of mitochondrial cytochrome c binding to bacterial photosynthetic reaction center, reconstituted in liposome membranes and immobilized by detergent dialysis and avidin-biotin binding. Anal Chem 280 94-102, 2000. 

Haneskog, L., Zeng, C.-M., Lundqvist, A., and Lundahl, P, Biomembrane affinity chromatographic analysis of inhibitor binding to the human red cell nucleoside transporter in immobilized cells, vesicles and proteoliposomes. Biochim. Biophys. Acta, 1371, 1-4, 1998. 

Hage, D.S., Walters, R.R., and Hethcote, H.W., Split-peak affinity chromatographic studies of the immobilization-dependent adsorption kinetics of protein A, Anal. Chem., 58, 274-279, 1986. 

Huang, P.Y. and Carbonell, R.G., Affinity chromatographic screening of soluble combinatorial peptide libraries, Biotechnol. Bioeng., 63, 633-641, 2000. 

High-Performance Metal Ion Affinity Chromatographic Separation of... 

The immobilized ligand is an essential factor that determines the success of an affinity chromatographic method [12]. The method which is used for affinity ligand immobilization is important because actual or apparent activity of the final column can be affected. Decrease in... 

Extensive descriptions of affinity chromatographic techniques with protocob and recipes. 

Kumar A, Bansal V, Andersson J, Roychoudhury PK, Mattiasson B (2006), Super-macroporous cryogel matrix for integrated protein isolation - immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line, J. Chromatogr. A 1103 35-42. 

A major advance in the elimination of catalytic site inhomogeneity in new preparations came with the development of an affinity chromatographic method (65) for purifying the enzyme. This method made use of the known high affinity of xanthine oxidase for alloxanthine when the enzyme is in a fully reduced state. By attaching alloxanthine to a polymeric matrix, a selective absorption of active enzyme was achieved. 

Displacement chromatography has also been carried out in dye affinity chromatographic systems for the purification of lactate dehydrogenase (LDH).59 In that study, polyethyleneimine (PEI) was employed as a displacer on dye affinity matrices prepared by immobilizing Cibacron Blue 3GA or Procion Red HE-3B. 

Nevens, J. R., Mallia, A. K., Wendt, M. W., and Smith, P. K. (1992). Affinity chromatographic purification of immunoglobulin M antibodies utilizing immobilized mannan binding protein. ]. Chromatogr. 597, 247-256. 

Production, Export and Affinity Chromatographic Purification of Recombinant... 

Table 1 Comparison of the recombinant production and affinity chromatographic purification of GFP from B. megaterium [30]. Different affinity tag fusion forms of GFP were produced in II. megaterium WH323. Purification was performed using affinity chromatography. Amounts of purified GFP-Strep were determined using a Bradford protein assay kit (Bio-Rad Munich Germany) and BSA (Perbio Rockford USA) as standard. Amounts of purified GFP-His, His-TEV-GFP, Strep-Xa-GFP and Strep-TEV-GFP were calculated via their relative fluorescence per mg protein...

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