Elution profile chromatographic, protein

Fig. 10.9. Separation, purification, and complementation of fragments elastase T (16-125) and elastase T (126-245). Elution profile of proteins from a Sephadex G 100 column (a) N-acetyl [ C]elastase after limited proteolysis in a column equilibrated in 6 M GuHCl (100 mM acetate buffer, pH 5.0) (b) Purification of protein fragments (obtained as illustrated in a) by repeated chromatographic runs in the same conditions (c) Reassociation of fragments obtained in b. After incubation at pH 8, the proteins were fractionated in the column without GuHCl, at pH 8.0 (borate buffer, 50 mM), The arrows indicate the position corresponding to the elution of the intact protein (from Ghelis et al, 1978).

Fig. 2 Chromatographic comparison of protein elution profiles from PEZ and ZrP04 phases. Particles (4.5 pm) were packed in 50 mm X 4.6 mm i.d. eolumns. Mobile phase 30 min linear gradient from 50 to 500 mM potassium phosphate dibasic, pH 7.0 0.5 mL/min ambient temperature deteetion at 280 nm. Peaks 1—myoglobin, 2—lysozyme, 3— R-chymotrypsin, 4— ribonuclease A, 5—cytochrome c. (From Ref. [17].)...

The CCC fractions, HDL-LDL and VLDL-serum proteins, were each separately dialyzed against distilled water until the concentration of the potassium phosphate was decreased to that in the starting buffer used for the hydroxyapatite chromatography. These two fractions were concentrated separately by ultrafiltration. The concentrates of both fractions were chromatographed on the hydroxyapatite column. Fig. 4 shows the elution profile on hydroxyapatite obtained from the HDL-LDL fraction. A 1.4-mL volume of the concentrate was loaded onto a Bio-Gel HTP DNA-grade column (5.0 x 2.5 cm I.D.)... 

Fig 2-Elution profile from a DEAE cellulose column(cm 2 X 30).Elution buffer 10 mM Tris-Cl,pH 8 500 mg proteins. Specific activity= 4moles " C02/hr/g. Insert=chromatographic pattern of active fraction on cellulose thin layer developped in butanol-pyridine -acetic acid-water(45 30 9 36)and sprayed with ninhydrin. 

Gradient elution was carried out with a mixture of two solvents solvent A consisted of 0.1% TFA in water and solvent B consisted of 0.1% TFA in 80% (v/v) aqueous acetonitrile. Proteins were eluted with a series of linear gradients, increasing the proportion of solvent B from 36% to 56% over 20 min, from 56% to 60% of B over 10 min, finishing with 60-36% of B in 5 min. The flow rate was 0.5 ml/min, the column temperature was 45 0.1 °C, and the detection was performed at a wavelength of 215 nm. Different chromatographic profiles were obtained for each type of milk in binary mixtures, as shown in Fig. 6. 

---