Hydroxyapatite columns

The enzyme was purified to 277 fold with very high specific activity, 1387 unit/mg protein by hydroxyapatite column chromatography (Figure 3). 

Figure 3. The profile of pectinase on Figure 4. The profile of pectinase on hydroxyapatite column chromatograph Toyopearl 55HW gel filtration column size 1x60 mm, 10-500 mM phosphate size 1.1x55 cm. flow rate 15 ml/h, 50 buffer pH 5.8, flow rate 12 ml/h mM acetate buffer pH 5.25...

Various chromatographic techniques may be utilized to purify urokinase further. Commonly employed methods include anion-exchange (DEAE-based) chromatography, gel filtration on Sephadex G-100 and chromatography on hydroxyapatite columns. Urokinase is a relatively stable molecule. It remains active subsequent to incubation at 60 °C for several hours, or brief incubation at pHs as low as 1.0 or as high as 10.0. 

Narita H, Takeda Y, Takagaki K, Nakamura T, Harata S, Endo M (1995) Identification of glycosaminoglycans using high-performance liquid chromatography on a hydroxyapatite column. Anal Biochem 232 133-136... 

Red-cell (dark adapted) Blepharisma japonicum were cultured in Pisa, in the dark, at 23 °C, in the presence of the Enterobacter aerogenes bacterium as food supply [7]. Blue-cell (light adapted) Blepharisma japonicum were produced by in vivo photoconversion of blepharismin into oxyblepharismin under a low intensity cold white lamp (below 10 W/m2). Blue cells were washed, collected by low speed centrifugation and resuspended in a 20-mM sodium cholate solution. The chromoprotein was obtained by FPLC chromatography of this preparation, on a hydroxyapatite column. The applied eluent was a phosphate buffer (pH 7.4), first 0.05 M and then 0.2 M. This ionic strength step affects the affinity of the biomolecules with the hydroxyapatite [8]. 

Methods that rely on specific interaction between plasmid DNA and a solid support. Examples are adsorption to nitrocellulose microfilters and hydroxyapatite columns. 

R Kasai, H Yamaguchi, O Tanaka. High performance liquid chromatography of glycosides on a new type of hydroxyapatite column. J Chromatogr 407 205 -210, 1987. 

Disorders in lipoprotein metabolism are critical in the etiology of several disease states, such as coronary heart disease and atherosclerosis. Thus, there is considerable interest in the development of novel methods for the analysis of lipoprotein complexes. A simple chromatographic method for the separation of high-density lipoprotein (HDL), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) from intact serum or plasma has been reported recently [65]. The separation was achieved by using an hydroxyapatite column and elution with pH 7.4 phosphate buffer with lOOpl injections of whole... 

Bemardi, G., Chromatography of nucleic acids on hydroxyapatite columns, Methods Enzymol., 21D, 95, 1971. 

K21. Kostner, G. M., and Holasek, A., The separation of human serum high density lipoproteins by hydroxyapatite column chromatography. Biochim. Biophys. Acta 488, 417-431 (1977). 

Figure 9. Radiochemical and protein analysis of column fractions after hydroxyapatite column chromatography of plasmin-treated (3-casein for various reaction times , total activity or specific activity, Fraction 1 M, total activity, Fraction 2 , specific activity, residual /3-casein O, total protein, Fraction 1. Values for total protein and total activity have been adjusted to correspond to trie same amount of total protein (21.92 mg) applied to each column (28).

FIGURE 13 Separation of mouse IgG I monoclonal antibody from ascitic fluid on a hydroxyapatite column (HA Ultrogel). Column l.l i.d.X 10 cm height initial buffer 10 mM phosphate, pH 6.8 sample 16 mL of ascitic fluid previously filtered elutions 50 m M phosphate buffer pH 6.8 (a), 100 m M phosphate buffer, pH 6.8 (b), 200 mM phosphate buffer, pH 6.8 (c). Flow rate 20 cm / hr regeneration I M sodium hydroxide. Antibodies are collected in buffer b purity by SDS - PAGE is 83%, yield is 77%. 

Bernardi, G., Giro, M. G., and Gaillard, C. (1972). Chromatography of polypeptides and proteins on hydroxyapatite columns Some new developments. Biochim. Biophys. Acta 278, 409-420. 

Purification of Monoclonal Antibody. Immunoglobulins were precipitated from the pooled ascites by addition of an equal volume of saturated ammonium sulfate [50% (NH4)2S04]. The precipitate was collected by centrifugation (20 min 10,240 X g), dissolved in 0.01 M sodium phosphate (pH 6.8), and reprecipitated. After the second ammonium sulfate precipitation, the pellet was dissolved in a minimum volume of 0.01 M sodium phosphate (pH 6.8) and centrifuged for 10 min at 10,600 X g). The resulting supemate was applied to a P6G, gel filtration polyacrylamid, column (Bio-gel Biorad, Rockville Center, NY 1.5 X 40 cm). Fractions containing protein were pooled and applied to a hydroxyapatite column that had been equilibrated with 0.01 M sodium phosphate (pH 6.8). Proteins were eluted with a linear gradient of 0.01 to 0.3 M sodium phosphate. 

Gorbunoff, M.J. (1990) Protein chromatography on hydroxyapatite columns. Methods Enzymd. 182, 329-339. 

An Fe-S protein has been found to be present also in the b /f complex of chloroplasts and cyanobacteria. This protein has been dissociated from the complex with Triton X-100 and hydroxyapatite column chromatography, and was shown to be associated with the 20000 Da subunit in chloroplasts [111] and the 22000 Da one in A. variabilis [128]. In all cases the dissociation resulted in an irreversible loss of activity the involvement of the Fe-S protein in electron transport was also proved by the inhibition by an antibody raised against the Triton isolated protein (but not by one against the SDS denatured subunit) [129,130]. An oxidoreduction potential of 0.290 V was measured in intact chloroplast membranes and in the complex or in the isolated homogeneous preparation the potential was pH independent below pH 8 [111]. 

The first peak of enzyme solution was dialyzed against 10 mM sodium succinate buffer, pH 6.0, without EDTA and with 2-mercaptoethanol, then applied to a hydroxyapatite column equilibrated with the same buffer. Stepwise elution with ammonium sulfate in this buffer yielded a single peak in 0.1 M ammonium sulfate fraction. The pooled enzyme was concentrated with 0.8 saturated ammonium sulfate to give 1% protein concentration in imidazole-glycylglycine buffer, pH 7.1. Addition of ammonium sulfate to between 0.5 and 0.55 saturation yielded a turbid solution which in turn yielded crystals after 24 hours at 4 C. The second peak was applied to a Sephadex G-200 column equilibrated with 0.01 M... 

After this incubation the solution is added to hydroxyapatite (column or centrifuge tube) usually at (Tjn— 15)°C and the unreasso-ciated, single-stranded, DNA is eluted with 0.12 M phosphate buffer. Reassociated, double-stranded, DNA is eluted with 0.30 M phosphate buffer. 

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.)... 

More recently, it was demonstrated that the thermistor approach could be used to monitor specific interactions of fluoride ions with silica-packed columns in the flow injection mode. A thermometric method for detection of fluoride [56] was developed that relies on the specific interaction of fluoride with hydroxyapatite. The detection principle is based on the measurement of the enthalpy change upon adsorption of fluoride onto ceramic hydroxyapatite, by temperature monitoring with a thermistor-based flow injection calorimeter. The detection limit for fluoride was 0.1 ppm, which is in the same range as that of a commercial ion-selective electrode. The method could be applied to fluoride in aqueous solution as well as in cosmetic preparations. The system yielded highly reproducible results over at least 6 months, without the need of replacing or regenerating the ceramic hydroxyapatite column. The ease of operation of thermal sensing and the ability to couple the system to flow injection analysis provided a versatile, low-cost, and rapid detection method for fluoride. 

After concentration, the solubilized enzyme was applied to a gel filtration column. The enzymes from pear and tomato were purified further (1.5-3 fold) by isoelectric focusing (20,25). Chloro-plastic hydroperoxide lyase was solubilized from tea leaves with Tween 20 and partially purified 8.5-fold with 34% recovery by hydroxyapatite column chromatography (33). Although attempts at... 

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