Hydroxyapatite separation method

ADVANTAGES AND DISADVANTAGES OF SOLUTION HYBRIDIZATION AND HYDROXYAPATITE SEPARATION COMPARED WITH OTHER DNA DNA HYBRIDIZATION METHODS... 

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... 

Aoyama, K., and Chiba, J. (1993). Separation of different molecular forms of mouse IgA and IgM monoclonal antibodies by high-performance liquid chromatography on spherical hydroxyapatite beads. J. Immunol. Methods 162, 201-210. 

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). 

Separation for measurement The basis of measurement depends on the ability to separate the unbound or free" Ag from the Ab-Ag complex, which itself relies on differences in properties between the two components while maintaining (that is not disrupting) the Ab-Ag complex (Figure 10.6). There are a number of reagents used for separation including charcoal, hydroxyapatite, ammonium sulfate and polyethylene glycol, but the method that will be considered here relies on the use of dextran-coated activated charcoal (see Figure 10.7). With inherent... 

The use of hydroxyapatite for DNA-fractionation exploits either the process of DNA denaturation or DNA reassociation. DNA denat-uration is the separation of the two complementary strands of native DNA by heat or agents like formamide or other methods. Denaturation takes place more easily the higher the A-I-T content of the DNA. Reassociation is the forming of a complementary double helix from single strands of DNA. This depends mainly on the concentrations of the complementary strands and hence on the repetition frequency of the sequence involved (Britten and Kohne 1966, 1968). 

Hydroxyapatite (HA) chromatography is a typical I didn t know what else to do method and thus most purifiers use it as the last step. HA chromatography is an inexpensive and easy method that can process large amounts of protein. However, the purification factors are often bad (2 to 5) and the yields moderate (40 to 60%). With acidic proteins, HA sometimes exhibits stunning purification effects. This is especially true for preparations that are prepurified via lEC, because HA separates acidic proteins by principles other than an ion exchanger. HA is a calcium phosphate mineral that binds proteins via two mechanisms (Figure 5.5). 

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. 

To separate the various tRNAs, numerous methods—including chromatography (on resins, silicic acid, hydroxyapatite, Sephadex, etc.), electrophoresis, and counter current—have been devised. An interesting procedure consists of treating the amino acid-charged RNA with either periodate or converting the... 

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