Protein sieving

Despite some refinements in the methods, the basic principles and protocols of gel electrophoresis have not changed appreciably since their introduction. Proteins are introduced into a gel matrix and separated by the combined effects of an electrical field, buffer ions, and the gel itself, which acts as a protein sieve. At the completion of the electrophoresis run, separated proteins in the gel are stained to make them visible, then analyzed qualitatively or quantitatively. The topic has been covered in numerous texts, methods articles, and reviews.1-11 In addition, apparatus and reagents for analytical and preparative gel electrophoresis are available from several suppliers. 

Norden AGW, Lapsley M, Lee PJ, Pusey CD, Scheinman SJ, Tam FWK, Thakker RV, Unwin RJ, Worng O. Glomerular protein sieving and implications for renal failure in Fanconi syndrome. Kidney Int 2001 60 1885-1892. 

Mehta and Zydney [41] show a similar relationship exists for ultrafiltration membranes where transport through the membrane occurs by convective pore flow. A Robeson plot was created by taking the selectivity of an ultrafiltration membrane as the reciprocal of the protein sieving coefficient (the ratio of protein concentration in the permeate to that in the fluid adjacent to the membrane surface) and the permeability as the solvent hydraulic permeability. A plot of literature data for bovine serum albumin separation shows the existence of an upper bound. The location of the upper bound was predicted assuming the... 

Second, most membrane materials adsorb proteins. Worse, the adsorption is membrane-material specific and is dependent on concentration, pH, ionic strength, temperature, and so on. Adsorption has two consequences it changes the membrane pore size because solutes are adsorbed near and in membrane pores and it removes protein from the permeate by adsorption in addition to that removed by sieving. Porter (op. cit., p. 160) gives an illustrative table for adsorption of Cytochrome C on materials used for UF membranes, with values ranging from 1 to 25 percent. Because of the adsorption effects, membranes are characterized only when clean. Fouling has a dramatic effect on membrane retention, as is explained in its own section below. 

Product recoveiy from reversed micellar solutions can often be attained by simple back extrac tion, by contacting with an aqueous solution having salt concentration and pH that disfavors protein solu-bihzation, but this is not always a reliable method. Addition of cosolvents such as ethyl acetate or alcohols can lead to a disruption of the micelles and expulsion of the protein species, but this may also lead to protein denaturation. These additives must be removed by distillation, for example, to enable reconstitution of the micellar phase. Temperature increases can similarly lead to product release as a concentrated aqueous solution. Removal of the water from the reversed micelles by molecular sieves or sihca gel has also been found to cause a precipitation of the protein from the organic phase. 

In reconstitution experiments, the self-assembly of the pore-forming protein a-hemolysin of Staphylococcus aureus (aHL) [181-183] was examined in plain and S-layer-supported lipid bilayers. Staphylococcal aHL formed lytic pores when added to the lipid-exposed side of the DPhPC bilayer with or without an attached S-layer from B coagulans E38/vl. The assembly of aHL pores was slower at S-layer-supported compared to unsupported folded membranes. No assembly could be detected upon adding aHL monomers to the S-layer face of the composite membrane. Therefore, the intrinsic molecular sieving properties of the S-layer lattice did not allow passage of aHL monomers through the S-layer pores to the lipid bilayer [142]. 

Henn, SW Ackers, GK, Molecular Sieve Studies of Interacting Protein Systems, The Journal of Biological Chemistry 244, 465, 1969. 

Dry bean curd refuse was used as the substrate in the lactic acid fermentation with simultaneous saccharification (SSF). The dry bean curd refuse was preliminarily sieved under a mesh size of 250 II m. It contained 12.3% water, 4.0% ash, 0.8% lipid, 29.3% protein, 53.6% carbohydrate, respectively, in weight basis. The cellulase derived from Aspergilltis niger with an enzymatic activity of 25,000 units/g (Tokyo Kasei Industry Inc.) was employed as the saccharification enzyme. 

Protein recovery via disruption has also been achieved by adsorbing water from the w/o-ME solution, which causes protein to precipitate out of solution. Methods of water removal include adsorption using silica gel [73,151], molecular sieves [152], or salt crystals [58,163], or formation of clanthrate hydrates [154]. In most of the cases reported, the released protein appeared as a solid phase that, importantly, was virtually surfactant-free. In contrast to the dilution technique, it appears that dehydration more successfully released biomolecules that are hydrophilic rather than hydrophobic. 

Hjerten, S. and Mosbach, R., Molecular sieve chromatography of proteins on columns of cross-linked polyacrylamide, Anal. Biochem., 3, 109, 1962. 

Capillary SDS-Sieving Electrophoresis In the presence of a sieving matrix, mobility decreases monotonically with molecular weight for SDS-complexed proteins. This relationship is the basis of SDS-PAGE separation of proteins. 

Widhalm et al. (1991) reported the use of noncrosslinked polyacrylamide for protein separation in fused silica capillaries. This matrix has low viscosity and can be replaced between separations, greatly facilitating automation of the separation. A wide range of noncrosslinked polymers has been used for size-based protein separations. Noncrosslinked polymers do not form a gel, and it is inappropriate to refer to this separation as gel electrophoresis. A number of names have been used for the method. In an effort to standardize nomenclature, IUPAC has used the term capillary sieving electrophoresis. 

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