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). 

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. 

Figure 10.2 Optimization of a purification protocol by LC-MS analysis. (1) LC-MS analysis of C-terminal fragment from reference molecule purified by cation-exchange chromatography (CEX) and hydroxyapatite chromatography (HA). (2) Incubation at 37°C under acidic conditions shows degradation of the purified molecule if CEX precedes HA. (3) The molecule is stabilized when the sequence of the two purification steps is swapped. 

Hydroxyapatite chromatography, separating molecules by differential surface binding to phosphate and calcium sites on a microcrystalline matrix of hydroxyapatite ([Ca5 (P04) 3OH] 2)... 

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 7. PAGE of reaction mixture after plasmin hydrolysis of B-casein for 0, 20, 30, 40, and 70 min (Slots 1-5) and Fractions 1 ana 2 from hydroxyapatite chromatography of hydrolysis products after plasmin treatment of fi-casein for 70 min (Slots 6 and 7). Fraction 2 contains a further phosphopeptide that is not visible in Slot 7 but appears on disc gels in the expected position for proteose peptone component 8F (cf. Ref. 32) (28).

Figure 8. Hydroxyapatite chromatography of plasmin-treated 6-casein. The P-casein (110 mg) containing 1.62 fiCi M-P-C was incubated with plasmin and one-fifth of the reaction mixture was withdrawn at t — 0, 20, 30, 40 and 70 min. Samples were dissolved in 2.0-mL column buffer (lOmM Na2HPOi-NaHsPOi, pH 6.8, containing 6M urea) and a known amount (90-100% of each) was applied to columns of hydroxyapatite (1.6 X 10 cm) equilibrated with the same buffer. Columns were eluted at a flow rate of 20 mL/h with column buffer (40 mL) followed by 300 mM Na HPOi,-NaH2POi pH 6.8, containing 6M urea (110 mL) 5.0 mL fractions were collected. The profile shown is for a reaction time of 40 min (28).

Yamasaki, Y., Yokoyama, A., Ohnaka, A., Kato, Y., Murotsu, T., and Matsubara, K.-I. (1989). High-performance hydroxyapatite chromatography of nucleic acids. ]. Chromatogr. 467, 299-303. 

Presence of charged clusters Hydroxyapatite chromatography Mediated by phosphate-rich regions... 

Alkaline elution techniques In one type of method the cellular DNA is denatured in alkali followed by hydroxyapatite chromatography which separates single stranded DNA from double stranded DNA (Britten and Kohne, 1965). The production of single-stranded DNA is related to the number of SSBs in the DNA which act as unwinding points. As an example the effects of UV-irradiation on the integrity of mammalian cell DNA has been studied using this approach (Collins, 1977). Initially mammalian cells in culture are extensively labelled with [3H]thymidine (see Adams, this series, 1980). 

After dilution to 20 ml with distilled water lysates can be stored overnight at 4°C before hydroxyapatite chromatography. 

Two monoclonal antibodies, one highly specific for human albumin (HSA-1) and one of broader specificity for several primates (HSA-2), were selected for subsequent use. These antibodies were purified by hydroxyapatite chromatography to single heavy- and light-chain bands (Figure 4) and then biotinylated. 

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. 

By means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, and hydroxyapatite chromatography we succeeded in isolating and purifying two NADPH-dependent oxidoreductases from enzyme extracts of Saccharomvces cerevisiae. which catalyze the enantioselective reduction of 3-oxoacid esters to (S)- and (R)-3-hydroxyacid esters (11). 

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. 4 Stepwise elution profile of HDLs and LDLs by hydroxyapatite chromatography. Column Bio-Gel HTP DNA-grade hydroxyapatite (5.0 x 2.5 cm I.D.) eluents 75 and 290 mM potassium phosphate buffers at pH 7.4 flow rate 1.0 mL/ min sample 1.4 mL concentrated HDL-LDL CCC fraction.

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