Dextran blue

Amphoteric hydrophobic Blue dextran, collagen, gelatin, hydrophobic proteins Hydrophobic peptides Buffer or salt solution with organic solvent (e,g, 20% CH3CN in 0.1 M NaNOi) 35-45% CH3CN in 0.1% TFA... 

FIGURE 16.5 Broad standard calibration (linear mode) of a semipreparative Sephacryl S-IOOO system (95 x 1.6 cm) with an aqueous mixture of Blue Dextran, Dextran T-SOO, and glucose eluent 0.005 M NaOH V, i = 75 ml. = 162 ml. 

The selective separation range of P-6/S-200 was determined with Blue Dextran (Vexdi exclusion limit) and fructose (V,o total permeation limit). Molecular weight (degree of polymerization) calibration (Fig. 16.22) was established with dextran standards and low dp pullulans (dp 3, 6, 9, 12, 15, 18) formed by the controlled hydrolysis of high dp pullulan. 

Chromatorapf c methods For gel filtration of polysaccharide fraction PI, a Sephacryl S-300 chromatographyc column (1,1 X 46,7 cm) was calibrated with standard dextrans (molecular mass range 266, 72, 40, and 17 KDa Sigma Chemicals), and the void volume determined with blue dextran. Polysaccharide sample (0.5 mL 2 mg/mL) was applied and eluted with 50 mM NaOH, fractions 1 mL being collected and carbohydrate absorbance (phenol-H2S04) being monitored. 

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]... 

Gel Permeation Chromatography. Samples were filtered on columns of Bio-Gel P-6DG (Bio-Rad Laboratories), and columns of Sephadex G-10, G-25, and G-50 (Pharmacia Corp.) using deionized water as eluant. Gel filtration properties are expressed in terms of the distribution coefficient calculated from the relationship = (V - V )/ (V - V ) where the the volume at which a component elutes, is the void volume, and is the total volume of the system. Blue dextran 2,000 and xylose were used to determine and Vj respectively. 

Fig. 20. Structured flows developed between the bottom solution containing dextran with 6.146 x 10-2 g/g phosphate buffer at pH 6.0 and dextranase with 10 units/g phosphate buffer (= 2.63 x 10s g/g phosphate buffer) and the top solution containing dextran with 3.952 x 10-2 g/g phosphate buffer (including blue dextran). The boundary was formed 3 min after the dextranase was added to the dextran solution. The photo was taken 30 min after formation of the initial boundary531...

The column is calibrated using proteins of known molar mass. The relative retention volumes 0.0 and 1.0 are defined by the elution of Blue Dextran (molecular weight 2 000 000) and sulfosalicylic acid (molecular weight 218), respectively. 

With 0.5M sodium hydroxide as eluent, Sephadex G-50 effects fractionation in the molar mass range 1000-15000 dalton and can be used for a period of 3-4 weeks with a single calibration carried out with proteins and polypeptides of known molar mass, as revealed by Figure 10. Relative retention volumes 0.0 and 1.0 are defined with Blue Dextran and phenol, respectively. 

Figure 2. Gel permeation chromatography on Sephadex G-75 of 1, blue dextran and phenol standard 2, aquatic river humus 3, CFH 4, chlorinated aquatic river humus and 5, organic compounds from drinking water (Philadelphia I, no. 9).

Void volume is measured by passing a large, inert molecule through the column." Its elution volume is defined as F0. Blue Dextran 2000, a blue dye of molecular mass 2 X 106, is commonly used for this purpose. The volume Vm can be calculated from the measured column bed volume per gram of dry gel. For example, 1 g of dry Sephadex G-100 produces 15 to 20 mL of bed volume when swollen with aqueous solution. The solid phase occupies only 1 mL of the bed volume, so Vm is 14 to 19 mL, or 93-95% of the total column volume. Different solid phases produce widely varying column bed volumes when swollen with solvent. 

B. Blue Dextran 2000 was eluted during gel filtration in a volume of 36.4 mL from a 2.0 X 40 cm (diameter X length) column of Sephadex G-50, which fractionates molecules in the molecular mass range 1 500 to 30 000. 

Figure 3-9 Elution volume of various proteins on a column of Sephadex G-200 as a function of molecular mass. The right-hand vertical axis shows the ratio of the elution volumes to that of blue dextran, a high-molecular-mass polysaccharide that is excluded from the internal volume. After Andrews.193...

Figure 6. Elution profile obtained on examination of the gel permeation behavior of 14C acetylated S. aureus Factor III. Cytochrome-c and fluorescein labeled E. coli HPr were included in the applied sample as internal standards. Absorbance at 410 nm (cytochrome-c), fluorescein fluorescence (HPr), and radioactivity (Factor III) are plotted vs. fraction number. Inset shows the elution profiles for the external (blue dextran) and internal (fiuoroglycine) column markers. Data in this figure are from unpublished work (20).

Void Volume This is the total space surrounding the gel particles in a packed column. This value is determined by measuring the volume of solvent required to elute a solute that is completely excluded from the gel matrix. Most columns can be calibrated for void volume with a dye, blue dextran, which has an average molecular mass of2,000,000 daltons. 

Kav is the availability constant of a substance with molecular weight M, Ve is the elution volume, Vt is the total volume of the column, V0 is the void volume or the interstitial space between the beads in the bed. The void volume could not be determined by the elution volume of Blue Dextran 2000 (Pharmacia Fine Chemicals AB, Uppsala, Sweden, Mw about two million) since it would have separated in the column. We used a 0.5% lipopolysaccharide solution (Bacto Lipopolysaccharide W, Salmonella typhosa 0901 ref. nr. 3124-25, Difco Laboratories, Detroit, MI, USA) with a molecular weight of above 150,000,000, which is excluded by the gel. 

Zinc-binding components of urine were examined using modified gel chromatography (15). Urine (3 ml) was chromatographed on Sephadex G-25 columns (2.5 x 40 cm) equilibrated with a buffer containing 10 ppm Zn as Zn(N03)2 and 10 mM Tris buffer, pH 7.4. Fractions of 3 ml were analyzed by atomic absorption spectroscopy. Void volume of the column was determined with blue dextran. 

To improve light collection for absorbance detection, a photosensor array was integrated on an acrylic chip with a smooth and transparent surface. This allows absorbance measurement (420-1000 nm) down to 0.0004 AU. Detection of 5 mg/ml blue dextran and 10 mg/mL each of lactic dehydrogenase (39 kDa) and P-galactosidase (116 kDa) has been carried out [240]. 

The flow cell mixing characteristics were investigated by alternating solutions of the polysaccharide blue dextran with CBS alone and measuring the light transmission through the cell as a function of time. Also the volume of the pump chamber was determined to be 1.78 cm3 using this technique. 

Figure 3. Light transmission through flow cell (no membrane in place) during wash-in and wash-out of a blue dextran solution at 2.24 mL/min.

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