Gel filtration of protein

Fig. 4-2 Gel filtration of proteins. Elution volumes Vcl are plotted for a series of proteins of different Mr applied to a column of Sephadex G-200. The two data points denoted by were not used in constructing the calibration curve.

Wl. Ward, D. N., and Amott, M. S., Gel filtration of proteins, with particular reference to the glycoprotein, luteinizing hormone. Anal. Biochem. 12, 296-302 (1965). 

These materials are used for the different modes of biochromatographic separation, ion exchange, hydrophobic interaction, gel filtration, and also affinity chromatography. Some materials are underivatized but not commercially available. In general, resins suitable for gel filtration of proteins are good candidates for affinity chromatography. 

Analytical gel filtration on Sephadex G-lOO gave symmetric elution curves of the absorbance at 278 nm and 417 nm, and of the protein determined by the Lowry method (Fig. 14). Calibrating the elution volume by gel filtration of proteins of known molecular weights, a molecular weight of 58,000 was obtained for cytochrome b. 

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). 

Purified LBP is obtained from the crude LBP separated in the gel filtration of the 35 kDa luciferase on Sephadex G-100 (see Fig. 8.2). The fractions of crude LBP are combined and the protein is precipitated with ammonium sulfate (75% saturation). The precipitate is dissolved in a small volume of lOmM Tris-HCl/5 mM 2-mercaptoethanol, pH 8, and a small amount of luciferin is added as a tracer. Then, the crude LBP is purified on a column of Sephadex G-200 (Hastings and Dunlap, 1986). The fractions of LBP are identified by luminescence produced by the addition of luciferase at pH 6.3 the luminescence due to the tracer luciferin is proportional to the amount of LBP in each fraction. 

Curculin which is extracted with 0.5 M sodium chloride from the fruits of Curculigo latifolia and purified by ammonium sulphate fractionation, CM-sepharose ion-exchange chromatography and gel filtration.The protein acts as a low calorie sweetener and has a maximum sweetness equal to 0.35 M of sucrose. In addition to its sweetness, curculin also has taste modifying abilities since water and sour substances elicit a sweet taste after consumption of curculin. Currently, there is no other protein that has both sweet taste and taste modifying abilities. 

Figure 3.21. Gel filtration of total colostrum whey protein on Sephadex G-200. Column 120 cm length, 3.7 cm diameter gel volume, 1420 cm3, 21.6 ml/hr. (From Mack et al. 1969. Reprinted with permission of AVI Publishing Co., Westport, Conn.)...

Acid phosphatase of S. aureus PS 55 is eluted from the cell surface by 1.0 M KC1 at pH 8.5. Gel filtration of this material gave a 44-fold purification. The protein seems homogeneous by gel filtration, starch block electrophoresis, and analytical ultracentrifugation with the weight of approximately 58,000 (12a). 

Gel filtration of crude cellulase on Sephadex G-75 gave one cellobiase, a major HMW endoglucanase, a LMW endoglucanase, and one cellobiohydrolase (Figure 8). The purified enzyme and the crude enzyme protein was analyzed by SDS-gel electrophoresis and resulted in the gels shown in Figure 9. 

Figure 7. Sephadex G-100 gel filtration of cellobiohydrolase. (-O-) Protein (A280 wn), (- -) activity with respect to Avicel.

For insertion into premade doxorubicin-loaded liposomes (Doxil), mix equal volumes of liposomes and lipidation reaction mixture and incubate at 37°C for 12-16 h (see Note 7). Purification of unreacted lipid and protein is not necessary at this step, because they do not interfere with insertion process and will be removed eventually by gel-filtration of decorated liposomes on Sepharose CL-4B. 

Figure 4.2 Gel filtration of a large-molecular-weight protein (solid line) and a small-molecular-weight protein (dotted line). The latter penetrates the beads and takes a longer route through the column. The large-molecular-weight protein passes among the beads, taking a shorter route through the column.

Figure 4.3 Gel filtration of a set of standard proteins on BioRad Biogel A 1.5 M column. The y scale is logarithmic. The x scale is the ratio of protein eluted volume to void volume. Point 2 is thyroglobulin (MW 670,000), 3 is bovine IgG (MW 158,000), 4 is ovalbumin (MW 44,000), 5 is myoglobin (MW 17,000), and 6 is cyanocobalamin (vitamin B12, MW 1350). (Reproduced by permission of BioRad Laboratories.)...

A specific exopolyphosphatase was tightly bound to the mitochondrial membranes of S. cerevisiae (Lichko et al, 1998). This was the first known example of membrane-bound exopolyphosphatases. It was characterized by its higher activity with PolyPs of greater chain lengths (Table 6.6). Under gel filtration of a solubilized preparation of mitochondrial membranes, this activity was shown to be associated with proteins of 76 and 140 kDa. A special feature of this exopolyphosphatase was its inhibition by divalent metal cations (Table 6.5). 

Figure 2-4 Gel filtration of excluded, partially included, and fully included proteins. (Partially included,...

Table II. Amino acid composition1 of glutenin and the protein fractions obtained from gel-filtration of reduced and alkylated glutenin (see Fig. 5)...

In gel filtration, a protein mixture (the mobile phase) is applied to a column of small beads with pores of carefully controlled size (the stationary phase). The movement of the solute is dependent on the flow of the mobile phase and the Brownian motion of the solute molecules, causing their diffusion into and out of the chromatographic bed. Large proteins, above the exclusion limit of the gel, cannot enter the pores and are hence eluted in the void volume of the column. Small proteins enter the pores and are therefore eluted in the total volume" of the column and intermediate size proteins are eluted between the void and total volumes. Proteins are therefore eluted in order of decreasing molecular size. 

EN91 Dasgupta, A. and Dean, R. (1992). Gel filtration of uremic sera only partially improves the protein binding of phenytoin. Clin. Chem. 38, 1004, Abstr. 296. 

An alternative to dialysis methods is the gel filtration technique of Hummel and Dreyer (57), as modified by Price (58). A column of Sephadex G-25 is equilibrated with a buffer containing a desired concentration of calcium and 45CaCl2, and is used for gel filtration of the binding protein. As the process of gel filtration proceeds, the protein migrates in the excluded volume of the column, removing calcium ions from the column buffer until equilibrium is reached. The protein peak, in the void volume of the column, contains above-base line amounts of calcium. The amount of calcium bound to the protein can be found by dividing the molar concentration of calcium above the base line value by the molar protein concentration. A separate gel filtration run is used to determine each point on the binding plot. This is at once time-consum-... 

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