Fractionation methods electrophoresis

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. 

Fairly wide use has been made of preparative gel electrophoresis in protein chemistry, and in principle there is no reason why the same procedures should not be adopted for use with nucleic acids which have the advantage that much may be accomplished with very small quantities of purified material. Thus, it is relatively easy in many situations to introduce radioactive label at very high levels and specific activity, and the use of for this purpose offers a degree of sensitivity that cannot be matched in work on proteins. The extinction coefficients of nucleic acids are also very high in the ultraviolet, so that with say 20 pg in 1 ml or less it is possible to measure optical properties, thermal melting profiles, sedimentation coefficients, and even molecular weights by sedimentation equilibrium in an instrument equipped with scanner optics. Consequently, the sacrifice of resolution that, by a malign law of nature, always accompanies any attempt to scale up an analytical fractionation method is often at least partly avoided. 

Work on the metabolism of the nucleic acids has been of central interest in many areas of molecular biology, and the diversity of the applications of gel electrophoresis in this sphere no doubt reflects the very wide interest that attaches to it, as well as the advantages inherent in the method. Essentially the desired attributes of a fractionation method in such work are high resolution, rapidity, economy in terms... 

As an alternative to transfer methods, gels in which nucleic acid is fractionated can, after drying, be submitted directly to denaturation and hybridization within the gel. Finally, fractionation by electrophoresis after a solution hybridization step (Section 12.4) or after PCR and in-gel detection is also convenient. A tracer amount of labeled precursor can be added to the PCR mix (Verbeek and Tijssen, 1991) or a 5 -labeled internal primer can be added at the end, followed by 1 PCR cycle (Parker and Burmer, 1991) and the products analyzed in the gel. Drying of the gel enhances the signal. 

In proteome studies, the process usually begins with a highly complex mixture of proteins, typically hundreds of unknown species. This requires one or several protein fractionation steps such as 1-D or 2-D gel electrophoresis or LC separation of the proteins and peptides prior to MALDI-MS analysis. The different types of fractionation methods can be combined in several ways for example, SDS-PAGE followed by LC separation of in-gel-digested peptides. In contrast to ESI, LC-separation techniques cannot be directly coupled to the MALDI instruments. Instead, the LC-separated peptide or protein fractions are spotted onto the MALDI-target, and then analyzed in an offline approach. 

Fig. 9.2 Relative concentrations of P450 in human liver microsomes. a P450s in 60 liver samples were estimated using immunochemical methods (electrophoresis/immunoblot-ting) [52]. Because of cross-reactivity, the individual P450s in subfamilies are not distinguished. The unknown fraction is the difference between the sum of the immunochemi-cally determined forms and the total amount, calculated from Fe -CO versus Fe " difference spectroscopy [53]. b-d Estimates were made using liquid chromatography-mass spectrometry (LC-MS) proteomic analysis with heavy-atom peptides, b Results of an analysis of 50 pooled human liver samples (XenoTech, HLM610 preparation) [54]. c Results reported in the same reference as Part 5 [54] as means from analysis often individual human samples, d Analysis of a pooled set of 23 human liver samples by another laboratory [55]...

Whether DLS, DWS, Mie scattering, or other applications in which unfractionated samples are analyzed, the resulting distributions produced by modern instruments, while frequently facile to obtain and neat in appearance, must be treated with caution, as there is usually a large amount of data smoothing, fitting, and assumptions applied in using inverse Laplace transform and several other commonly employed methods. The best means of finding distributions of size and mass continue to be fractionation methods, such as SEC [32-34], field flow fractionation (FEE) [35-37], capillary electrophoresis [38], capillary hydrodynamic fractionation [39], and so on. 

One of the earliest methods of detecting hyaluronic acid was the formation of a so-called mucin clot in presence of proteins, by addition of acetic acid to fluids or tissue extracts. Detection by mecisurements of the decrease of viscosity, after addition of hyaluronidase, is not reliable, because the pure enz)nne is not available, and the mixture of enzymes used also degrades the chondroitin sulfates. Definite identification by chemical means requires separation on a microscale, using selective adsorption on a column, or fractionation by electrophoresis, determination of the ph3 cal characteristics, and analysis of the components. 

Albumin. Investigation iato the safety of bovine plasma for clinical use was undertaken ia the eady 1940s ia anticipation of wartime need (26). Using modem proteia chemistry methods, including electrophoresis and ultracentrifugation, it was shown that most of the human adverse reactions to blood substitutes were caused by the globulin fraction and that albumin was safe for parenteral use. Human albumin is now used extensively as a plasma expander ia many clinical settings. 

Electroosmotic flow in a capillary also makes it possible to analyze both cations and anions in the same sample. The only requirement is that the electroosmotic flow downstream is of a greater magnitude than electrophoresis of the oppositely charged ions upstream. Electro osmosis is the preferred method of generating flow in the capillary, because the variation in the flow profile occurs within a fraction of Kr from the wall (49). When electro osmosis is used for sample injection, differing amounts of analyte can be found between the sample in the capillary and the uninjected sample, because of different electrophoretic mobilities of analytes (50). Two other methods of generating flow are with gravity or with a pump. 

Stalcup aiid co-workers [14] adapted this method to a continuous elution mini-prep electrophoresis apparatus shown in Fig. 11-3. In this apparatus, the end of the electrophoretic gel is continuously washed with elution buffer. The eluent can then be monitored using an HPLC detector (Fig. 11-4) and sent to a fraction collector where the purified enantiomers, as well as the chiral additive, may be recovered. In this system, the gel configuration was approximately 100 mm x 7 mm, and was aircooled. The number of theoretical plates obtained for 0.5 mg of piperoxan with this gel was approximately 200. A larger, water-cooled gel was able to handle 15 mg of... 

Because of the instability of many of the compounds involved, it is necessary to determine the chemical recoveries in all cases. This requires the use of macro quantities (10 mg up to several hundred mg) of carriers and target compounds. This, in turn, makes it impractical to use the various thin-layer methods, such as paper and thin-layer chromatography and paper electrophoresis, although such methods have proved useful in identifying products and in checking the purity of fractions. The separation methods now most commonly used are column chromatography and sublimation. 

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