Proteins free-flow electrophoresis

Douglas et al. [98] have measured protein (serum albumin, ovalbumin, and hemoglobin) mobilities over a range of pH values using a free-flow electrophoresis apparatus and a particle electrophoresis apparatus. They found good agreement between the two measurements however, they also found some differences between their measurements and those reported in the older literature. They attributed the differences to the use of moving-boundary electrophoresis methods in the early experimental work and to differences in... 

Kuhn, R. and Wagner, H., Application of free flow electrophoresis to the preparative purification of basic proteins from an E. coli cell extract,... 

Hoffmann, R, Ji, H., Moritz, R. L., Connolly, L. M., Frecklington, D. F., Layton, M. J., Eddes, J. S., Simpson, R. J. (2001). Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215 a non two-dimensional gel electrophoresis-based proteome analysis strategy. Proteomics 1(7), 807. 

Moritz, R.L., Clippingdale, A.B., Kapp, E.A., Eddes, J.S., Ji, H., Gilbert, S., Connolly, L.M. and Simpson, R.J. (2005) Application of 2-D free-flow electrophoresis/RP-HPLC for proteomic analysis of human plasma depleted of multi high-abundance proteins. Proteomics 5, 3402-3413. 

The technique of choice in the detection and isolation of protein complexes is immunoprecipitation [15], whereas in some cases centrifugation or free-flow electrophoresis can be applied for the purification of cellular organelles. Immunoprecipitation permits the purification of specific proteins against which an antibody has been raised. Affinity-based methods have been developed for a variety of organelles. When apphed to the isolation of protein complexes, the antibody should target one of the proteins in the complex. The antibody-protein complex precipitates from solution. Alternatively, inunobilization of the primary antibody to agarose (beads) can be applied. Mrrlti-step procedttres have been developed as well. 

As a result of experiments originally conducted on Apollo 16 (34), commercial electrophoretic efforts have been undertaken on space shuttle flights (45,46). The apparatus involved employs free flow electrophoresis of a protein mixture for the purification and recovery of a very high valued product. The process itself is proprietary. However, data and models are available from earlier flights and other experiments (34,47,48). 

Methods for cell and sub-cellular fractionation include immuno-isolation, electromigration e.g. free flow electrophoresis), flow cytometiy, density gradient isolation of organelles and sequential extraction. The use of some of these approaches to investigate the mitochondrial and myofibrillar sub-proteomes of the heart will be described in Sections 3.4 and 3.6 respectively. A particular problem in proteomic analysis of the heart is the diversity of cell types that are present. Proteomic profiles of total myocardial lysates are dominated by the proteins present in cardiac myocytes, but such samples... 

Further purification of this fraction is possible by a second (continuous) gradient centrifugation [64]. Alternatively, free-flow electrophoresis in the presence of Mg-ATP has been applied [63], which leads to a fraction with an activity of 110 mol/mg protein/h in the presence of valinomycin. These latter purification steps often lead to a reduced recovery, and also to a decrease or loss of the effect of ionophores on the activity, indicating that the vesicular structures have been opened by the purification procedure. Less purified preparations have, therefore, mostly been used for the study of vesicular transport. 

Free-Flow Electrophoresis of Protein Samples To reduce the dynamic range of proteins to be analyzed, and subsequently to maximize the number of... 

Unfortunately, it is not yet possible to purify informosomes free of contaminating ribosomes, subribosomal particles, and soluble proteins of the cytoplasm. The only useful purification method is centrifugation in a CsCl density gradient, but this requires a prefixation with formaldehyde or with other cross-linking agents (Spirin et al., 1965). The fixation makes it impossible to study adequately the proteins of particles or their activities. Another possible approach is to use free flow electrophoresis, which should differentiate ribosomes and D-RNP, but such work has only just started (Schweiger and Hannig, 1970). 

A very important question concerns the nature of the proteins of polysomal mRNP. This question has not yet been answered because of the difficulties in the isolation of polysomal mRNPs. Since reticulocyte mRNPs can now be obtained in a pure state, the question may be answered in the near future. In another approach, Schweiger and Hannig (1970) tried to find proteins typical of nuclear D-RNP by direct analysis of polysomes. They separated the total polysomal protein into several fractions using free flow electrophoresis and then compared the fractions with nuclear D-RNPs obtained by means of polyacrylamide gel electrophoresis. In free polysomes and in membrane-bound polysomes, proteins similar to proteins of the nuclear D-RNP were found. If the membrane-bound polysomes are separated from the membranes by treatment with pure deoxycholate, these proteins are dissociated from polysomes and thus resemble the proteins of nuclear D-RNP in their sensitivity to deoxycholate. 

Thus, in general, the question about the similarity of the proteins composing the nuclear and polysomal particles remains open pending the isolation of pure polysomal mRNP. Free flow electrophoresis may be a good approach since, according to preliminary data by Schweiger and Hannig (1970), it permits the separation of ribosomes and mRNP. 

ID Separation of Proteins There has been considerable research devoted to tbe development of nricioflu-idic platforms capable of performing small-scale protein separations. In addition to diffusion based methods [3], nearly every type of electrokineticaUy driven separation has been demonstrated in a cbip-based platform, including free-flow electrophoresis, capillary electrophoresis, capillary gel electrophoresis, isoelectric focusing (lEF), micellar electrokinetic chromatography and capillary electrochemical chromatography. 

Braun RJ, Kinkl N, Zischka H, Ueffing M 2009. 16-BAC/SDS-PAGE analysis of membrane proteins of yeast mitochondria purified by free flow electrophoresis. Methods Mol Biol 528 83-107. 

Electrophoretic methods are widely used alternatives for the analytical determination of the enantiomeric purity of chiral compounds [194]. Due to the high elTi-ciency of capillary electrophoresis, separations can be achieved even when very low selectivities are observed. At a preparative scale, these methods are well established for the purification of proteins and cells [195] but there is very little published on enantioselective separations. Only recently, some interest in chiral preparative applications has been manifested. Separation of the enantiomers ofterbu-taline [196] and piperoxan [197] have been reported by classical gel electrophoresis using sulfated cyclodextrin as a chiral additive, while the separation of the enantiomers of methadone could be successfully achieved by using free-fluid isotachophoresis [198] and by applying a process called interval-flow electrophoresis [199]. 

Wagner, H. Kessler, R. "Free-flow field-step focusing a new method for preparative protein isolation" In Electrophoresis 83. Stathakos, D., Ed. W. de Gruyter Berlin, Germany, 1983, pp. 303-312. 

Song Y-A et al (2010) Free-flow ztme electrophoresis of peptides and proteins in PDMS microchip fOT narrow pi range sample prefractionation coupled with mass spectrometry. Anal Chem 82(6) 2317-2325... 

The theory of electrophoresis has been adequately covered in the excellent textbooks of Giddings [1] and Andrews [2] as well as in specific manuals [3], [4]. For discussion on electrophoresis in free liquid media, e.g., curtain, freeflow, endless belt, field-flow-fractionation, particle, and cell electrophoresis the reader is referred to a comprehensive review by Van Oss [5] and to a book largely devoted to continuous-flow electrophoresis [6], Here the focus is mostly on electrophoresis in a capillary support, i.e, in gel-stabilized media, and discussion is limited to protein applications. 

Fig. 4. Massive production of green fluorescent protein (GFP) by the continuous-flow cell-free method. Sodium dodecyl sulfide-polyacrylamide gel electrophoresis analysis of GFP produced during 14 d of reaction. mRNA produced by transcription of circular plasmid of Ehime University (pEU) was used for the translation reaction in the dialysis membrane system and was added every 48 h. A 0.1 -pL aliquot of the mixture was run on the gel and protein bands were stained with Coomassie Brilliant Blue. The arrow shows GFP and st designates an authentic GFP band (0.5 pg).

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