Proteins homogenates

Poehling reported a microscale two-dimensional gel electrophoresis system 25 years ago (Poehling and Neuhogg, 1980 Neuhoff, 2000). In that system, separation in the IEF dimension was performed in thin, gel-filled tubes. After IEF, the gel was transferred to a 3 cm x 3.5 cm polyacrylamide gel, where proteins were separated by SDS-PAGE. Several hundred components were resolved from a few micrograms of protein homogenate. 

CSE and Micellar Electrokinetic Capillary Chromatogarphy (MECC) of Complex Protein Homogenates... 

FIGURE 15.1 One-dimensional capillary electrophoresis separation of a protein homogenate prepared from the hTERT cell line. Both separations were preformed in 30 pm ID, 145 pm OD, 20 cm long capillaries at 20,000 V. (a) Micellar electrokinetic chromatography performed with a 100 mM CHES, 100 mM Tris, and 15 mM SDS buffer at pH 8.7. Sample is electro-kinetically injected with 0.25 kV for 1 s (b) Capillary sieving electrophoresis performed in 5% Dextran (513 kDa), 100 mM CHES, 100 mM Tris, 3.5 mM SDS, pH 8.7. 

FIGURE 15.4 Computer record of a two-dimensional capillary electrophoresis analysis of a protein homogenate prepared from ahiopsy obtained from the fundus of a Barrett s esophagus patient. The data were generated hy performing 1 s transfers between capillaries and a 9 s second-dimension separation. The first-dimension separation employed the same buffer as the CSE separation in Fig. 15.1 and the second-dimension separation employed the same buffer as the MECC separation in Fig. 15.1. 

Michels, D.A., Hu, S., Damhrowitz, K.A., Eggertson, M.J., Lauterhach, K., Dovichi, NJ. (2004). Capillary sieving electrophoresis-micellar electrokinetic chromatography fully automated two-dimensional capillary electrophoresis analysis of Deinococcus radiodurans protein homogenate. Electrophoresis 25, 3098-3105. 

The third test of protein homogeneity, developments from which remain in common use, was that of electrophoresis. Arne Tiselius had been a research assistant in Svedberg s laboratory. From 1925 he pioneered the application of electrophoresis to the analysis and separation of protein mixtures, showing with dialyzed serum differences in mobility of the protein components and the presence of three classes of globulins, a, B, and y. 

Only a few bacterial and viral sialidases have been purified to high purity or even to protein homogeneity."0 Complete purification of sia-lidase on a preparative scale from the culture filtrate ofC. perfringens was achieved111 by using poly(acrylamide) gel-electrophoresis as the final purification step (see Section VI,1). It is necessary that such purified sialidases be available, as the presence of proteases or other gly-cosidases in the enzyme preparations would lead to severe errors, not only in studies of substrate specificity, but also in cell biological and medical studies (see Sections VI and VII). 

Perez-Juan, M., Flores, M., and Toldra, F. (2006). Model studies on the efficacy of protein homogenates from raw pork muscle and dry-cured ham in binding selected flavor compounds. J. Agric. Food Chem. 54,4802 808. 

The metal is firmly associated with the protein moiety of the enzyme. Consequently, the metalloenzyme can be isolated from its matrix, retaining all of its metal complement in the natural state (Edsall, 1951). During purification, the ratio of metal to enzyme-protein increases. With complete purification, a protein homogeneous by physical-chemical criteria is obtained, and the ratio of metal to protein becomes constant (Keilin and Mann, 1940a). 

Chen YT, Stockert E, Jungbluth A, et al. Serological analysis of Melan-A (MART-1), a melanocyte-specific protein homogeneously expressed in human melanomas. Proc Natl Acad Sci USA. 1996 93 5916-5919. 

CRP. C-Reactive Protein. Homogeneous acute phase protein, found in man and most other animals. Discovered as a result of its precipitation with C-polysaccharide (CPS) in sera nf patients with infections and inflammatory disease W. S. Tillet, T, Francis, J. Exp. Med. 52, 561... 

Herriott, R. M. The Solubility Diagram as a Criterion of Protein Homogeneity, in Methods in Enzymol-ogy4. 212 (1957). 

Mione, M. C., Danevic, C., Boardman, P, Harris, B., and Pamavelas, J. G. (1994) Lineage analysis reveals neurotransmitter (GABA or glutamate) but not calciumbinding protein homogeneity in clonaUy related cortical neurons. J. Neurosci. 14, 107-123. 

Example. Determination of glycine in hydrolyzed protein [98] 152.6 mg hydrolyzed protein homogenized with 5.07 mg C-labeled glycine of specific activity U =(96.2 1.2) counts min mg" (relative activities are. sufficient). After separation of a portion of glycine from the mixture. 

Although the encephalitogenic effect of brain or spinal cord homogenate had been known for many years, it was the separation of two proteins, homogeneous by ultracentri-fugational and electrophoretic criteria, that promoted further research. These were a collagen-like protein with low EAE activity [124], and a basic protein obtained by a different... 

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