Proteins hydroxyapatite chromatography

Final alcohol precipitation not only allows for removal of the phenol and any remaining non-covalently bound hydrocarbon but also concentrates the DNA. Ribonuclease treatment removes any contaminating RNA. Additional purification by cesium chloride centrifugation (35) is also often performed. This is particularly suited to small quantities of DNA. Hydroxyapatite chromatography is also effective in separating RNA, proteins, and DNA (36.37). 

Hydroxyapatite occurs naturally as a mineral in phosphate rock and also constitutes the mineral portion of bone. It may also be used to fractionate protein by chromatography. 

Dong M, Baggetto LG, Folson P, LeMaire M, Penin F. Complete removal and exchange of sodium dodecyl sulfate bound to soluble and membrane proteins and restoration of their activities, using ceramic hydroxyapatite chromatography. Anal Biochem 1997 247 333-341. 

M Fountoulakis, M-F Takacs, P Berndt, H Langen, B Takacs. Enrichment of low abundance proteins of Escherichia coli by hydroxyapatite chromatography. Electrophoresis 20 2181-2195, 1999. 

Figure 4. SDS-PAGE profile of monoclonal antibodies HSA-1 and HSA-2. After purification by hydroxyapatite chromatography, HSA-1 (lane 2) and HSA-2 (lane 3) were electrophoresed on a 10% polyacrylamide gel in the presence of 0.1% SDS. Approximately 1 (xg of HSA-1 and HSA-2 was loaded in each lane. The gel shows two peptide bands corresponding to heavy and light (56-and 24-kilodalton [KD], respectively) immunoglobulin chains and no evidence of other contaminating proteins. Lane 1 was loaded with molecular weight markers.

Renault, F., Chabriere, E., Andrieu, J.P., Dublet, B., Masson, P., Rochu, D. (2006). Tandem purification of two HDL-associated partner proteins in human plasma, paraoxonase (PONl) and phosphate binding protein (HPBP) using hydroxyapatite chromatography. J. Chromatogr. B 836 15-21. 

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. 5 Stepwise elution profile of serum proteins and VLDLs by hydroxyapatite chromatography. Column Bio-Gel HTP DNA-grade hydroxyapatite (5.0 x 2.5 cm I.D.) eluents 290 and 650 mM potassium phosphate buffers at pH 7.4 flow rate 1.0 mL/min sample 1.5 mL concentrated VLDL-serum protein CCC fraction.

Kato, Y., Nakamura, K., and Hashimoto, T. (1987) High-performance hydroxyapatite chromatography of proteins. 

CCC is a very useful technique for the separation and fractionation of human semm lipoproteins. The HDL-LDL and VLDL-serum protein fractions were directly obtained from human serum using a solvent system composed of 16% PEG 1000-12.5% potassium phosphate at pH 9.2. The separated HDL-LDL and VLDL-serum protein fractions were loaded onto hydroxyapatite columns and separated into HDLs, LDLs, VLDLs, and serum proteins, respectively. The combination of CCC and hydroxyapatite chromatography is a usefid method for the separation of the three main classes of lipoproteins from human seram, without prior ultracentrifugation. 

Hydroxyapatite columns have been shown to be helpiiil for the purification of proteins, especially when other types of chromatographic columns were not successful [12]. Although the separation mechanism of hydroxyapatite chromatography is not understood, it is known that it is not an ion-exchange mechanism. Applications of hydroxyapatite HPLC to the analysis of glycoproteins including... 

PEG based linear and dendritic polymer carrying end chelating groups (Mw 1000-50 000g/mol) Standard protein mixture/hydroxyapatite chromatography Smallest molecule best results [25[... 

The next step was to further isolate STE. This was accomplished by hydroxyapatite chromatography with a stepwise elution. The partially-purified STE almost exclusively hydrolyz stearoyl-ACP. The STE was purified from other proteins by FPLC Mono S. The active STE was eluted by a succinate buffer (20 mM, pH 4.5) which proved to be an efficient approach to obtain the high recovery. The STE was further purified by a Mono Q chromatography (Fig. 2) using Tris buffer (20 mM pH 8.5) with a 0 - 0.4 M NaCl gradient. The active fractions were then pooled and applied to an ACP-affinity column STE was eluted in two steps. 

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

Interest in the nature of interactions between shortchain organic surfactants and large molecular weight macromolecules and ions with hydroxyapatite extends to several fields. In the area of carles prevention and control, surfactant adsorption plays an important role in the Initial states of plaque formation (1-5) and in the adhesion of tooth restorative materials ( ). Interaction of hydroxyapatite with polypeptides in human urine is important in human biology as hydroxyapatite has been found as a major or minor component in a majority of kidney stones ( 7). Hydroxyapatite is used in column chromatography as a material for separating proteins (8-9). The flotation separation of apatite from... 

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