Separation of proteins

Since the total quantity (or activity) of a protein in a tissue sample is difficult to determine, the original (100%) quantity of protein at the start of a purification is usually based on measurements made on an aliquot of the initial homogenate. Each step in a purification process should remove extraneous protein and retain most of the protein of interest. A pure protein preparation is operationally defined as one that maintains a high activity per gram of protein following several purification steps, i.e., the optimum 

Many of the methods described earlier do not differentiate between different proteins but it is often necessary to determine the amount of one particular protein in the presence of others. Although proteins are composed of amino acids, the problems involved in the separation of individual proteins are considerably increased compared with the separation of amino acids, and the large relative molecular mass means that some of the simpler separation techniques such as 

Iso-electric focusing techniques probably give the best resolution and many of the resulting bands are due to specific proteins. They are used mainly as a qualitative or a semi-quantitative technique due primarily to the large number of bands that develop and are of particular value when successive samples from the same source need to be compared for the presence or absence of a particular protein or for investigation of physical properties, e.g. p/. 

It is important to realize that the use of these different media for the same sample will result in separation patterns that cannot be easily compared with one another. The separation of serum proteins on cellulose acetate will result in 5-7 bands, while the use of polyacrylamide gel will give 17 bands. 

In addition to its pH, the concentration of a buffer also affects the mobility of proteins. At high concentrations the zeta potential of the protein is reduced resulting in a shorter distance of migration. However, because higher concentrations of buffer give improved resolution, a compromise concentration has to be found and buffers with ionic strength (pi) varying from 0.025 to 0.075 are frequently used. 

The various types of capillary electrophoresis are performed either in free solution or in gels. The choice of method depends on the nature of the sample and the analytical objective but capillary gel electrophoresis, including iso-electric focusing and SDS electrophoresis, is particularly useful for protein applications. 

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Foams are used industrially and are important in rubber preparations (foamed-latex) and in fire fighting. The foam floats as a continuous layer across the burning surface, so preventing the evolution of inflammable vapours. Foams are also used in gas absorption and in the separation of proteins from biological fluids. See anti-foaming agents. 

Gel-permeation media are extremely versatile and may be used for separation of particles such as vimses (Fig. 11) as well as proteins (34). Separations of proteins and other particles having sizes equivalent to a molecular weight of 40 x 10 are possible using the agar-based Sepharose-type gel. This particular gel has a limited temperature range for operation, however. It melts upon heating to 40°C (34). 

Uraizee and Narsimhan [Sep. Sci. Technol., 30(6), 847 (1995)] have provided a model for the continuous separation of proteins from dilute solutions. Although their work is focused on protein separation, the model should find general application to other separations. 

Misunderstandings arise when membrane users assume that MWCO means what it seems to say. The definition implies that a 50 kD membrane will separate a 25 kD material from a 75 kD material. The rule of thumb is that the molecular mass must differ by a factor of ten for a good separation. Special techniques are used to permit the separation of proteins with much smaller mass ratio. 

The combination of diafiltration and batch concentration can be used to fractionate two macrosolutes whose retentions differ by as little as 0.2. It is possible in principle to achieve separations that are competitive with chromatography. When tanks and other equipment are considered, as well as the floor space they occupy, the economics of membrane separation of proteins may be attractive [R. van Reis, U.S. Patent 5,256,294 (1993)]. 

The separation of proteins and peptides mixtures is the objective of protein biochemisdy. Albumin (Mr 66 000) concentration in a biological fluid (seaim, urine or cerebrbrospinal fluid) is assayed as markers for a series disease, such as nephritic syndrome or chronic glomuleronephritis. In diabetic patients the progression of microalbuminuria is accompanied by an increase in urinary concentrations of human semm albumen. In normal the excretion of albumin is 20 (tg/ml, in pathology - 20-200 p.g/ml. 

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

Size exclusion was first noted in the late fifties when separations of proteins on columns packed with swollen maize starch were observed (Lindqvist and Storgards, 1955 Lathe and Ruthven, 1956). The run time was typically 48 hr. With the advent of a commercial material for size separation of molecules, a gel of cross-linked dextran, researchers were given a purposely made material for size exclusion, or gel filtration, of solutes as described in the classical work by Porath and Flodin (1959). The material, named Sephadex, was made available commercially by Pharmacia in 1959. This promoted a rapid development of the technique and it was soon applied to the separation of proteins and aqueous polymers. The work by Porath and Flodin promoted Moore (1964) to apply the technique to size separation, gel permeation chromatography of organic molecules on gels of lightly cross-linked polystyrene (i.e., Styragel). 

FIGURE 2.15 Influence of the pore size of Sephacryl HR on the separation of proteins of various molecular mass. The protein mixture is composed of ferritin, aldolase, ovalbumin, and chymotrypsinogen A. [Reproduced from Hagel et al. (1989), with permission.]... 

The size separation of proteins has been routinely called gel filtration because of the historic use of cross-linked gels for this application. Specially modified Zorbax PSM columns, Zorbax GF-250 and Zorbax GE-450, are used for separating proteins by size. These columns are packed with porous silica micro-... 

For the classical form of size exclusion chromatography in organic solvents, packings based on highly cross-linked styrene-divinylbenzene are used. For SEC of polar polymers using polar or aqueous solvents, packings based on a polar methacrylate polymer are used. Diol-derivatized silica is used for the separation of proteins and other polar polymers. The different packings will be discussed in sections dedicated to their different application areas. 

Figure 9.3 Schematic illustration of the electrophoretic transfer of proteins in the chromatophoresis process. After being eluted from the HPLC column, the proteins were reduced with /3-mercaptoethanol in the protein reaction system (PRS), and then deposited onto the polyacrylamide gradient gel. (PRC, protein reaction cocktail). Reprinted from Journal of Chromatography, 443, W. G. Button et al., Separation of proteins by reversed-phase Mgh-performance liquid cliromatography , pp 363-379, copyright 1988, with permission from Elsevier Science.

W. G. Button, K. D. Nugent, T. K. Slattery, B. R. Summers and L. R. Snyder, Separation of proteins by reversed-phase high-performance liquid chromatography , 7. Chromatogr. 443 363-379(1988). 

P. D. Grossman, J. C. Colburn, H. H. Lauer, R. G. Nielsen, R. M. Riggin, G. S. Sittampalam and E. C. Rickard, Application of free-solution capillary electrophoresis to the analytical scale separation of proteins and peptides . Anal. Chem. 61 1186-1194 (1989). 

The most reliable methods of the preparation of stable adsorbents involve, however, a covalent attachment of the polymeric stationary phases to the solid supporting material. In addition, the more diffuse interfaces formed in this case (see Sect. 2.2) are often favourable for the separation of proteins. 

A prehminary study of the use of larch AGs in aqueous two-phase systems [394] revealed that this polysaccharide provides a low-cost alternative to fractionated dextrans for use in aqueous two-phase, two-polymer systems with polyethylene glycol (PEG). The narrow molecular-weight distribution (Mw/Mn of 1-2) and low viscosity at high concentration of AG can be exploited for reproducible separations of proteins under a variety of conditions. The AG/PEG systems were used with success for batch extractive bioconversions of cornstarch to cyclodextrin and glucose. 

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) An electrophoretic technique used for the separation of proteins. 

Rossieretal. [332] usedUV excimer laser photoablation to cut channels 50 microns deep by 100 microns wide in laminated PET. These channels were filled with PA, and rapid separation of proteins by isoelectric focusing was demonstrated. 

P. Juo and G. Stotzky, Electrophoretic. separation of proteins from roots and root exudates, Canadian Journal of Botany 48 1 7i (1970). 

Figure 4.22 Separation of proteins on a small particle gel column. Column 60 x 0.75 cm. TSK-Gel 2000 SW mobile phase, 0.01 M phosphate buffer (pH 6.5) with 0.2 M sodium sulfate flow rate,...

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