Membrane brain proteins, sample

Guinea pig brain membranes, 0.5 mg of protein/sample, were incubated with 12 different concentrations of the compounds in the presence of receptor-specific radioligands at 25°C, in a final volume of 1 ml of 50 mM Tris-HCl, pH 7.5. Nonspecific binding was determined using 1 M naloxone. Data are the mean values S.E.M. from three experiments, performed in triplicate. 

Hertz L., Schousboe A., Boechler N, Mukerji S., and Fedoroff S (1978) Kinetic characteristics of the glutamate uptake into normal astrocytes in culture Neurochem Res 3, 1-14 Hertz L., Yu A, Svenneby G, Kvamme E., Fosmark H., and Schousboe A. (1980) Absence of preferential glutamine uptake into neurons. An indication of a net transfer of TCA constituents from nerve endings to astrocytes Neurosct Lett 16, 103-109 Hoffman E K., Simonsen L O, and Lambert I H (1984) Volume-induced increase of K" and Cr permeabilities in Ehrlich ascites tumor cells Role of internal calcium / Membrane Biol 78, 211-222 Holloway P. W (1973) A simple procedure for removal of triton X-100 from protein samples Anal Biochem 53, 30 308 Hosli E. and Hosli L. (1976) Autoradiographic studies on the uptake of [ H] noradrenaline and [ H] GABA in cultured rat cerebellum Exp Brain Res 26, 319-324... 

Fig. 7. Membranes and purified proteins contain multiple PTX substrates. SDS-PAGE was in 8% gels, 15.5 cm long with a linear 4-8 M urea gradient. Only sections of the autoradiogram with molecules of M, 35000-45000 are shown. Membrane samples, 1 p.g protein/lane brain Gj/G and hRBC G, (Gk), 20 ng/lane.

Soft ionization techniques such as electrospray ionization and matrix assisted laser desorption are now routinely used to determine the mass of large hydrophilic polymers like proteins (27). However, as is usual for the ionization process, the presence of sails and detergents, which is common for biological samples, can affect the process significantly. The use of the on-line capillary reversed-phase HPLC in combination of the electrospray mass spectrometer (LC/MS) has made it possible to analyze such samples directly (10,16, 28). When GAP-43 isolated from the membrane fractions of bovine brain was analyzed, a single major peak with a minor peak corresponding to a phosphorylated species was observed (Fig. la). To study the posttranslational modifications in detail, the protein was digested with specific proteases such as lysyl... 

The final resuspension should be in a volume calculated to give an appropriate protein concentration approx 100 mg of membrane protein is obtained for every gram of brain tissue prepared. After the final resuspension, a sample of the homogenate is taken for determination of protein content by Bradford or Lowry assays. 

FIGURE 2. Immunodetection of Na,K-ATPase alpha and alpha+ subunits in developing rat brain. 30 ug of crude membrane protein from littermates of different days during development were prepared and separated by 7.5% SDS-PAGE, blotted onto diazophenylthioether paper, and probed with antisera. A anti-guinea pig Na,K-ATPase antiserum diluted 1 100. B the paper from A was reincubated with anti-rat Na,K-ATPase antiserum diluted 1 400. C a mixture of both antisera was used to probe the samples of a second litter. Numbers represent days of gestation (18.5, 20.5) or days of age. K, 2 ug purified kidney Na,K-ATPase. 

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