Protein gel filtration

It has been found that the polymer surface having appropriately grafted nonionic, water-soluble polymer chains minimizes protein adsorption and cell adhesion. It should be noted that such minimum protein adsorption has been well known for a long time for hydrogels used for protein analysis such as polyacrylamine (PAAm) gel for electrophoresis, sephadex for protein gel filtration, and soft agar for cell culture. It is likely that the surface structure of these hydrogels resembles that of the grafted surface described above. 

Several methods are available for determining the molecular weight of proteins. Gel-filtration column chromatography, SDS-PAGE, and ultracentrifugation are among the most commonly used. 

Size differences between ligand and binding protein (gel filtrations columns or PEG precipitation)... 

L. Hagel, "Gel Filtration" in J-C. Janson and L. Ryden, ed.. Protein Purification Principles, High Resolution Methods, and Applications, VCH Pubhshers,... 

Pish protein concentrate and soy protein concentrate have been used to prepare a low phenylalanine, high tyrosine peptide for use with phenylketonuria patients (150). The process includes pepsin hydrolysis at pH 1.5 ptonase hydrolysis at pH 6.5 to Hberate aromatic amino acids gel filtration on Sephadex G-15 to remove aromatic amino acids incubation with papain and ethyl esters of L-tyrosine and L-tryptophan, ie, plastein synthesis and ultrafiltration (qv). The plastein has a bland taste and odor and does not contain free amino acids. Yields of 69.3 and 60.9% from PPG and soy protein concentrate, respectively, have been attained. 

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 2.4 Calibration curve of dextran on Sephacryi S-300 SF. Calibration curves were calculated from one chromatogram of a broad MWD reference sample using data for the molecular mass distribution as obtained by a calibrated gel filtration column ( , upper curve) and on-line MALLS ( ). The calibration curve was found useful for estimating the size of globular proteins. [Reproduced from Hagel et al. (1993), with permission.]... 

Sample load is primarily a concern in preparative gel filtration. In analytical applications the only precaution is to ascertain that the sample volume is sufficiently low as not to contribute to peak widths (and thus decrease the quality of the information) (Hagel, 1985). The concentration of the sample should not exceed 30 mg/ml for globular proteins or 5 mg/ml for polymers and DNA (Hagel and Janson, 1992). 

Gel filtration is very suitable for the purity check of protein preparations, especially if these have been purified by adsorptive techniques. It can be expected that high-resolution gel filtration columns will easily separate dimeric forms from monomeric forms to reveal heterogeneities of the preparations. However, a size difference of less than 20% will not result in total resolution of the peaks (although the chromatogram may be used for a qualitative judgment of the... 

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-... 

A new protein of unknown structure has been purified. Gel filtration chromatography reveals that the native protein has a molecular weight of 240,000. Chromatography in the presence of 6 M guanidine hydrochloride yields only a peak for a protein of M, 60,000. Chromatography in the presence of 6 M guanidine hydrochloride and 10 mM /3-mercaptoethanol yields peaks for proteins of M, 34,000 and 26,000. Explain what can be determined about the structure of this protein from these data. 

The protein can be further purified by hydrophobic interaction chromatography on a column of Butyl Sepharose 4 Fast Flow (Pharmacia elution with decreasing concentration of (NH4)2S04 starting at 1.5 M), and gel filtration on a column of Superdex 200 Prep (Pharmacia Inouye et al., 2000). 

Fig. 8.2 Gel filtration on a column of Sephadex G-100 at pH 8 (both panels) of the crude extract of Gonyaulax polyedra cells prepared at pH 8 (upper panel) and prepared at pH 6 (lower panel). The activities of the 35 kDa and 130 kDa luciferases are measured by the addition of an excess of luciferin at pH 6.3 ( ) or at pH 8(A). The activity of the luciferin-bound LBP (luciferin-binding protein) in the upper panel is measured after the addition of an excess of 35 kDa luciferase at pH 6.3 ( ). In the lower panel, the LBP activity can be obtained by the addition of an excess of luciferin at pH 8, followed by the removal of unbound luciferin with a small column of Sephadex G-25 before the luminescence assay of bound luciferin at pH 6.3 (see the Section 8.2.8). The Overlap in the upper panel is the light emission resulting from the mixing of an aliquot of the fractions with pH 6.3 buffer. From Fogel and Hastings, 1971, with permission from Elsevier.

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. 

Gel filtration on Sephadex G 25, G 50 or G 75 beads was just available. After standardization with peptides and proteins of known length, the molecular weight of the respective fraction could be determined. Also cyanogen bromide peptides of collagen chains were available in the later sixties, thus leading to a more consistent standardization15"17. ... 

An extract from the soluble stromal proteins of purified and intact spinach-leaf chloroplasts was prepared by lysis of the cells in buffer, centrifugation of the suspension of broken cells, and concentration of the supernatant with removal of insoluble material. This extract contained all of the enzymes involved in the condensation of the cyclic moieties of thiamine, thiazole, and pyramine. Thus, the synthesis of thiamine in this extract following the addition of pyramine and putative precursors was a proof that the system had the possibility of building the thiazole. It was found that L-tyrosine was the donor of the C-2 carbon atom of thiazole, as in E. coli. Also, as in E. coli cells, addition of 1 -deoxy-D-f/irco-pen-tulose permitted synthesis of the thiamine structure. The relevant enzymes were localized by gel filtration in a fraction covering the 50- to 350-kDa molecular-mass range. This fraction was able to catalyze the formation of the thiazole moiety of thiamine from 0.1 -mM 1-deoxy-D-t/ireo-pentulose at the rate of 220 pmol per mg of protein per hour, in the presence of ATP and Mg2+. 

Water soluble protein with a relative molecular mass of ca. 32600, which particularly contains copper and zinc bound like chelate (ca. 4 gram atoms) and has superoxide-dismutase-activity. It is isolated from bovine liver or from hemolyzed, plasma free erythrocytes obtained from bovine blood. Purification by manyfold fractionated precipitation and solvolyse methods and definitive separation of the residual foreign proteins by denaturizing heating of the orgotein concentrate in buffer solution to ca. 65-70 C and gel filtration and/or dialysis. 

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