Gel filtration GF

To achieve highest resolution the sample volume must not exceed 5% of the total column volume. Gel filtration is independent of sample concentration, although above 50 mg/ml protein viscosity effects may cause fingering. Extremely viscous samples should be diluted. 

Correct sample preparation ensures good resolution and extends the life of the column. Sample buffer composition does not directly affect resolution. During separation the sample buffer is exchanged with buffer in the column. Viscous samples, which could cause an increase in back pressure and affect column packing, should be diluted. Samples must be free from particulate matter, particularly when working with bead sizes of 34pm or less (see Chapter 8 for details of sample clarification procedures) 

Pre-packed columns ensure reproducible results and highest performance. 

In gel filtration good column packing is essential. The resolution between two separated zones increases as the square root of column length. The following guidelines apply  

Column dimensions =minimum 50 cm bed height (Sephacryl media) minimum 30 cm bed height (Superdex, Superose media) 

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The evolution of media covering aqueous and nonaqueous systems on the one hand and analytical as well as microscale and macroscale preparative applications on the other hand has resulted in an arbitrarily nomenclature within the field. Thus the current practice is to refer to the separation principle based on solute size as size exclusion chromatography (SEC) whereas the application in aqueous systems is traditionally referred to as gel filtration (GF) and the application in nonaqueous systems is designated gel-permeation... 

The classification of chromatography as gel permeation chromatography (GPC size-exclusion chromatography, SEC gel filtration, GF), ion exchange chromatography (lEC), hydrophobic interaction chromatography (HIC), and affinity chromatography (AC) is... 

Gel Permeation Chromatography (GPC Gel Filtration, GF Size-Exclusion Chromatography, SEC)... 

Gel filtration (GF) Size and shape High speed Low capacity High resolution + + + +... 

Size exclusion (SEC) or Gel filtration (GF) Particles of well-defined size, of different sized pores Aqueous buffer Desalting, buffer exchange Determination of molecular weight Final polishing... 

The application and preparation of afEnity resins was first described in a paper by Cuatrecasas in 1968 [1]. In this work, nearly aU features of this technique were explored. Affinity chromatography is the youngest of the four major purification methods used in biochromatography. In 1968, ion exchange (lEX), hydrophobic interaction (HIC), and gel filtration (GF) were already well established. What new opportunities in the purification of biomolecules were then added by affinity chromatography ... 

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-... 

Gel Filtration. The lyophilized protein was redissolved in 50 mM phosphate buffer, pH 7.4 0.15 m NaCl 0.013 % sodium azide and loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fi actions were collected. Fractions with pectin lyase activity were combined, dialyzed against distilled water and used in the next step. To estimate the molecular mass of PNL, the column was calibrated with standard proteins (Sigma MW-GF-70 Albumin, 66,000 Da Carbonic Anhidrase, 29,00 Cytochrome, 12,400 and Aprotinin, 6,500). The proteins were eluted in the conditions described above and their volumes (F ) were calculated fi om the peak maximum of the absorbance at 280 nm. The partition coefficient was obtained fi om the relationship where F, represents the bed volmne of column and F the void volume (which was calculated using blue dextran. Sigma). The molecular mass was determined using a standard curve of vs the logarithm of the molecular masses of the standards [28, 29]... 

IHI TABLE 13 Selection of Gel Filtration Media (GF) for the Separation of Antibodies and Related Molecules... 

GF is well suited for use after any of the concentrating techniques (IEX, HIC, AC, EBA) since the target protein will be eluted in a reduced volume and the components from the elution buffer will not affect the gel filtration separation (gel filtration is a non-binding technique with limited volume capacity and unaffected by buffer conditions). 

GF separates proteins with differences in molecular size. The technique is ideal for the final polishing steps in a purification when sample volumes have been reduced (sample volume significantly influences speed and resolution in gel filtration). Samples are eluted isocratically (single buffer, no gradient Figure 41). Buffer conditions are varied to suit the sample type or the requirements for further purification, analysis or storage step, since buffer composition does not directly affect resolution. Proteins are collected in purified form in the chosen buffer. 

Parameters such as molecular weight of target proteins and contaminants, resolution required, scale of purification should be considered when selecting gel filtration media. Figure 42 on page 89 shows a guide to selection of GF media. 

Dibutyl TeUurium (Hydrazine Method)1 To a stirred mixture of 0.40 g (10 mmol) powdered sodium hydroxide, 0.64 g (5.0 mmol) of finely ground tellurium powder, and 10 m/ dimethylformamide are added, dropwise by syringe under an atmosphere of nitrogen at 50- 60° 0.50 ml (7.1 mmol) 80% hydrazine hydrate. The mixture is stirred for 3 h. A solution of 1.4 g (10 mmol) bromobutane in 2 ml dimethylformamide is added and the mixture heated at 60° for 30 min, then cooled to 20°, and extracted with petroleum ether (30 -60°). The organic phase is separated, washed with water, and dried with anhydrous calcium chloride. The mixture is filtered and the solvent evaporated from the filtrate. The residue is purified by preparative TLC (silica gel, Merck 60 GF 254/hexane) yield 57% b.p. 111-114713 torr. 

The contribution of mucus carbon to Phaeocystis organic carbon can be assessed in three different ways. In the first, cells are mechanically separated from the mucus matrix by filtration through GF/C filters. Fractionation by filtration yields contributions of 5-80% of mucus carbon to POC (reviewed by Riegman and Van Boekel 1996). As many have pointed out, however, Phaeocystis cells are fragile and may be disrupted by filtration under pressure (Veldhuis and Admiraal 1985 Van Rijssel et al. 1997 Mathot et al. 2000). By releasing their water soluble carbon the fraction of mucus-carbon may be overestimated. On the other hand, mucopolysaccharides may form gels on the filter (Chin et al. 1998) as a result of which their contribution may be underestimated. Therefore, all studies in which colonies were fractionated by filtration or centrifugation have to be interpreted with care. 

Feed and product samples were analyzed using standard procedures. Molecular weight distribution were determined by gel permeation chromatography (Waters Associates). Sediment content in the liquid products was estimated by filtration through a glass fiber Whatman GF/A (1.6 pm porosity) filter at 100°C. 

In the assay procedure, labeled protein is isolated and separated from small labeled products by filtration. To each tube is added 14 ml of saturated ammonium sulfate solution, the top is replaced, and the tube is inverted. The contents are filtered under suction through a 24-mm glass fiber filter (Whatman GF/A) held in a polypropylene filter holder (Gel-man). The tubes, including the top, are washed twice with 15 ml of 50% saturated ammonium sulfate solution, and the washings are filtered. Each filter is washed three times with about 25 ml of 50% saturated ammonium sulfate solution, twice with 25 ml of ice-cold 1 N HCl, and twice with 15 ml of acetone the vacuum source is turned off while the funnel is being filled at each of these washing steps. The extensive wash procedure is required to obtain low blanks of the order of 500 cpm (0.05 to 0.1% of the total radioactivity in the sample filtered). At the conclusion of the washes, the filter is dry and free of ammonium sulfate and HCl. 

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