Fractionation methods proteins

The endothelin B receptor is an example of characterization of a homogeneous, affinity purified protein (Roos et al., 1998). Significant progress has been made in the development of techniques for more high-throughput identification of phosphorlyation events. Analysis of large sets of phosphorylated proteins is facilitated by the availability of affinity purification methods such as anti-phosphotyrosine or anti-phosphoserine antibodies or metal affinity chromatography (Neubauer and Mann, 1999 Soskic et al., 1999). These methods are not specific to a particular protein but rather are used to fractionate all proteins that are phosphorylated. 

The literature is devoid of any studies on the electrophoretic behavior of lignins. However, due to the presence of at least one phenolic hydroxyl group per lignin building unit, it appeared that such an investigation would be feasible. Moreover, due to the success of applying electrophoretic methods to the characterization and fractionation of proteins, a study as to the possible availability of the method to lignins was undertaken in this laboratory. 

To acquire information on the intrinsic metabolic activity of aquatic organisms, liver of carp (Cyprinus carpio Linnaeus), rainbow trout (Salmo gairdneri) and freshwater snail (Cipango-paludina japonica Martens) was dissected out, homogenized in 0.1M phosphate buffer, pH 7.5, and centrifuged at 105,000 g for 60 min to obtain the microsome-equivalent (described as the microsomal fraction hereafter) fraction. The protein content of microsomal and submicrosomal (supernatant fractions by Lowry s method, microsomal P-450 content ( ), activity of aniline hydroxylase (4) and aminopyrine N-demethylase (5) were determined. 

An alternative method which could be used to establish the fraction of protein that actually reaches the alveoli is the so-called co-aerosohzation. If a protein is aerosolized from a solution that also contains another low molecular weight substance (deposition marker), it can be assumed that the fractions of protein and deposition marker reaching the alveoli will be the same. The deposition marker should be a substance with a known alveolar epithelial membrane passage (e.g. tobramycin or a decapeptide) which does not undergo absorption after oral administration. The fraction of the deposition marker that is deposited in the alveoli can be established from plasma (and urine) measurements of the deposition marker. The maximum fraction of protein that can pass the alveolar membrane whl then be known. The ratio between the deposited fraction and the fraction that has been absorbed into the systemic circulation (as can be estabhshed form plasma or urine analysis) will provide an estimation of the protein passage across the alveolar membrane. 

Fractionation of proteins according to size utilizing cross-linked dextran or polyacrylamide gel columns was first demonstrated by Porath and Flodin 63 in 1959. This technique has become the most widely accepted method for separation and molecular weight determination of hydrophilic and some hydrophobic macromolecules using aqueous buffers with or without organic modifier. While this technique might not be unique in its ability to resolve and separate proteins, it is one additional simple and effective tool in the chemist s armamentarium. The theories behind size-exclusion HPLC and size-exclusion chromatography at low pressure are identical and are described in several publications. 31 34 36 39 44 64 65 ... 

After gross fractionation of proteins, as discussed above, more refined methods with greater resolution can be attempted. These methods, in order of... 

The purification of two proteins, UMP synthase from mammalian tumor cells, and lactose carrier protein from E. coli bacteria is described in detail to illustrate how different fractionation methods can be combined most effectively. 

L9. Levin, B., and Oberholzer, V. G., Paper electrophoresis of serum proteins with micro Kjeldahl nitrogen analysis of the protein fractions A comparison with free electrophoresis and salt fractionation methods. Am. J. Clin. Pathol. 23, 205 (1953). 

Halophilic enzymes are very unstable in low salt concentrations. Because some of the important fractionation methods in protein chemistry, such as electrophoresis or ion-exchange chromatography, cannot be applied at high salt concentrations, the available fractionation methods are rather limited. This basic difficulty is the main reason why the number of halophilic enzymes studied in pure form is very small. 

Fractionation of proteins by absorption and ion exchange. Part A, pp. 67-87 in A Laboratory Manual of Analytical Methods of Protein Chemistry, P. Alexander and R. J. Block, eds., Vol. 1, 254 pp., Pergamon Press, London, 1960. With S. Keller. 

This experiment employs a variety of fractionation methods to purify the bacterial DNA. Perchlorate ion is used to dissociate proteins from DNA. Chloroform-isoamyl alcohol is used to denature and precipitate proteins by lowering the di-... 

It had long been known that apoptosis and activation of caspase are coupled with mitochondrial permeabilization. However, until 1996, the molecular link between mitochondria and caspase activation had not been known. Dr. Wang s group at the University of Texas isolated activators of caspase 3 by using classical fractionation methods in 1996-1997. One protein that was required for activation of caspase 3 was a 15-kD protein with pink color, which was cytochrome c. This finding is rather surprising because cytochrome c is localized in mitochondria and has a well-established role as an electron carrier. Cytochrome c was later shown to be released... 

Once the membrane proteins have been solubilized, usually in a water-detergent medium, they must be fractionated. As each fractionation method brings its own constraints, this will further restrict the scope of possibilities that can be used for... 

For many years, the facility of the hydrochloric acid assay in gastric juice and the unavailability of good quantitative techniques for fractionation of proteins and mucosubstances have directed the interest of researchers toward the study of hydrochloric acid in the stomach. The only other material in gastric juice, studied and quantitated for many years and representative of the nondialyzable gastric secretory products, was pepsin. Study of other large molecular components has been hampered by the complexity of gastric juice, technical diflSculties encountered in its fractionation, and lack of adequate quantitative methods for the assay of these materials. 

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