Membrane immunoaffinity

Columns, dialysis membranes, capillaries or beads may be used in immunoaffinity application which is a non-covalent, irreversible purification process based on highly specific interactions between analyte and antibody [11]. 

We subsequently used extrinsically labeled diets for further fractionation by gel filtration and immunoaffinity chromatography. Mn in human milk was found to be predominantly (about 70%) bound to lactoferrin, the major iron-binding protein in human milk. Minor amounts were bound to casein in human milk and the milk fat globule membrane. It is therefore possible that changes in lactoferrin concentration in human milk may explain the developmental pattern... 

The composition of the lysis solution is dictated by the nature of the proteins under study and the subsequent techniques applied to the sample. One of the major choices to be made is whether or not a detergent is required at this stage. If the membrane and soluble fractions are to be separated the initial cell disruption protocol should not include a detergent, as many of the membrane proteins would be solubilized. In this case physical disruption of the cells should be used (e.g., sonication of cells or homogenization of tissues). The choice of lysis conditions is a vital consideration in this work, as proteins need to be solubilized while preservation of posttranslational modifications, inhibition of proteases, maintenance of protein-protein interactions, and, if an immunoaffinity purification step is to be performed, suitability for the antibody to function are essential. For example, SDS is very good at solubilizing membrane proteins but... 

Charcosset C, Su Z, Karoor S, Daun G, and Colton CK. Protein A immunoaffinity hollow fiber membranes for immunoglobuhn G purification Experimental characterization. Biotech. Bioeng. 1995 48 415 27. 

Platonova GA, Pankova GA, ITina lY, Vlasov GP, and Tennikova TB. Quantitative fast fractionation of a pool of polyclonal antibodies by immunoaffinity membrane chromatography. J. Chromatogr. A 1999 852 129-140. 

Nachman M, Azad ARM, and Bailon P. Efficient recovery of recombinant proteins using membrane-based immunoaffinity chromatography (MIC). Biotech. Bioeng. 1992 40 564-571. 

Using an LOD sensor, Bardeletti et al. (1986) achieved a linear concentration dependence for lactate between 0.25 and 250 pmolA. This unusually high sensitivity was obtained by immobilization of the enzyme on an immunoaffinity membrane such as is commercially available for antibody fixation (Pall Biodyne, USA). The large pores of this membrane provide very fast substrate and product diffusion. For LOD binding the membrane was simply dipped into an enzyme solution. 

Mascini and Mazzei (1986) succeeded in the covalent immobilization of PyOD to a Biodyne Immunoaffinity Membrane (Pall Biodyne, USA) containing carboxylic groups on the surface. The membrane was preactivated with a carbodiimide derivative. The enzyme membrane was fixed on the tip of a hydrogen peroxide-indicating Pt electrode between a cellulose acetate membrane and a further dialysis membrane. This pyruvate sensor has been applied to serum measurement. Over 30 days the sensitivity dropped by only 13%. 

McGillis, X, et al. (1987). Immunoaffinity Purification of Membrane Constituents of the IM-9 Lymphoblast Receptor for Substance P, Anal. Biochem. 164 502-513. 

Powell, C.E. Watson, C.S. Gametchu, B. Immunoaffinity isolation of native membrane glucocorticoid receptors from S-49 mGR-f+ cells biochemical characterization and interactions with hsp 70 and 90. Endocrine 1999,10, 271. 

Affinity chromatography is a separation procedure in which the protein to be separated is selectively adsorbed by an immobilized secondary molecule. Undoubtedly the most specific method used to purify membrane proteins at the moment is immunoaffinity chromatography. In this method, the specificity of an antibody directed against a protein is used as a tool in the purification. 

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