Isolation of membranes

Many important proteins (e.g., ion channels, neurotransmitter receptors, transporters, and ion pumps) are integral membrane proteins. You need membranes to examine the binding sites of these proteins. If appropriate to the question being researched, you can obtain the basic material (e.g., muscle, liver, brain) from the slaughterhouse. Pig brain is much less expensive than rat brain. 

The first step in obtaining membranes is the homogenization of the tissue. It is suspended in a homogenization buffer and then chopped up. The homogenization buffer is iso-osmotic to the tissue fluid, has a low ion strength, and contains cane sugar (250 to 320 mM), buffer (often 5 to 10 mM tris-Cl or Na-HEPES pH 7.4), and a mixture of different protease inhibitors (Bacitracin, PMSF, EDTA, and so on). But how do you chop it up  

The combined SI are centrifuged for 30 minute at 10,000 to 20,000 g to generate pellet P2 and supernatant S2. If you would like to remove the soluble proteins in the membrane vesicles, subject P2 to an osmotic shock. For this, P2 is resuspended in a tenfold volume of hb without sucrose and incubated for 30 minutes on ice, and the membranes are then centrifuged for 30 minutes at 20,000 g. The pellet is resuspended in buffer (e.g., 5 mM tris-Cl, pH 7.4), aliquoted, and frozen at -80 C. 

Plasma membranes from cell lines and cell cultures It is usually sufficient to scrape off the cells from the petri dish, to homogenize in PBS in the potter, and to make a P2 pellet from the homogenate. Finer methods appear in the following papers. 

(1981). Purification and Characterization of Human Lysosomes from EB-virus Transformed Lymphoblasts, Cell Res. 131 251-266. 

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Wagner, G.J. (1983). Higher plant vacuoles and tonoplasts. In Isolation of Membrane and Organelles from Plant Cells, ed. J.L. Hall and A.L. Moore, pp. 83-118. London Academic Press. 

As an example for combination of different methods in protein purification Fig. 3.2 gives the flow chart for the isolation of membrane protein complex. 

Harris, P. J. (1983). Cell walls. In J. L. Hall, A. L. Moore (Eds.), Isolation of membranes and organelles from plant cells (pp. 25-53). Aeademie Press, London. 

PBPs are membrane-bound enzymes. In general, PBPs, especially high-molecular-mass PBPs, consist of a short cytoplasmic domain in their N-terminus, followed by a hydrophobic transmembrane domain, then a transglycosylase domain (for class A PBPs) or a non-penicillin-binding domain (also called an unknown functional domain for class B PBPs), and finally a transpeptidase domain located in their C-terminus (Fig. 1 [1,2,15,56]). Thus, purification of PBPs traditionally involved the isolation of membranes and solubilization of PBPs from the membrane preparation by detergents. Since PBPs are present at a very low level (approximately 100-300 molecules per cell) [57-59] and also in multiple forms in the cell, this classical approach was not particularly efficient for producing a significant amount of a purified PBP protein of particular interest. 

Schagger, H. and von Jagow, G. (1991). Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. 

The control could be established if at any one time there was at the membrane more vesicles than those that were fusing. However in order to sustain the increase in secretion it would be necessary to transmit the signal back to the synthetic system and packaging process at the Golgi apparatus so that a new steady state system was obtained. This would produce the requisite number of vesicles containing the polysaccharides necessary to maintain the new rate of fusion and secretion. The turnover of the Golgi apparatus can be very fast in plant cells and times of 5-40 minutes have been calculated (36). In vitro experiments which involved isolation of membrane fractions from maize-root cells also showed that Ca was necessary for membrane fusion (37). Analysis of the membranes indicated that the Ca dependence involved membrane proteins and one of these was a Ca and Mg -dependent ATPase (38). 

Schagger, H., Cramer, W. A, and von Jagow, G. (1994). Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal. Biochem. 217, 220-230. 

It appeared, furthermore, that synaptosomal plasma membranes (SPMs) could easily be obtained after lysis and ftuther subcellular fractionation of such synaptosomal preparations. Essentially, this procedure permits isolation of synaptosomal plasma membranes with minimal contamination by glial cell elements. Throughout the years, this method has been modified and improved in many labs to permit detailed studies of neuronal signal transduction using isolated synaptosomal plasma membranes in vitro. Recently, synaptosomal plasma membranes have been used to study protein phosphorylation and dephosphorylation events (6-9), polyphosphomositide metabolism (10), and protein-protein interactions using chemical crosslinkers (9,11) and unmuno-precipitation techniques (6,12). Synaptosomal plasma membranes have also been used as source material for isolation of membrane proteins (13). 

Some precautions should be taken during the isolation of membrane-bound polysomes. For example, some D-RNPs, including nuclear 308 particles, are not stable and dissociate in the presence of the anionic detergent deoxycholate. The nonionic... 

HPSEC applied as a single technique for the isolation of membrane proteins will be useful only in special cases in which the desired protein has a large difference in molecular weight from the other components in a mixture, facilitating its separation from other proteins. HPSEC will be more useful in multidimensional chromatography when it is followed by one or more other modes of HPLC. 

HPIEC is a versatile technique for the isolation of membrane proteins and its possibilities are still largely unexplored. A wide variety of detergents, salts, and pH values can be employed to obtain a pure protein under physiological conditions. 

The incorporation of 3H-NeuNAc into endogenous acceptors of the two primary retina fractions is shown in Table 1. The labelling of lipids and proteins in total ROS free membranes was about six fold greater than the labelling of lipids and proteins found in total ROS membranes. Subfractionation of the ROS free membranes in a sucrose density gradient resulted in the isolation of membranes with different activities for the incorporation of NeuNAc. The highest activity was in subfraction Pla (Table 2). The incorporation of radioactivity into lipids paralleled the labelling of proteins in all the subfractions, from the more active Pla to the less active... 

Leegood, R.C., Walker, D.A., Chloroplasts, In JL Hall, AL Moore, eds. Isolation of Membranes and Organelles from Plant Cells Academic Press New York, 1983,... 

Growth conditions for cultivation of B. subtllls 168, Isolation of membranes and ultracytochemlcal methods have been described In our previous publications [5,6]. The ATPase activity was determined by the procedure of Clarke and Morris [7], protein was measured by the method of Lowry (1951) and SDS-gel electrophoresis was performed according to Laemmll [8]. 

For isolation of membranes, leaves were homogenized (blender) in a medium containing 0.3 M sucrose, 10 mM KCl, 1 mM MgCl2 50 mM Hepes, pH 7.5 (NaOH), filtered, centrifuged at 10 min at 6,000 Omax remove plastids and centrifuged 30 min at 60,000 gmax- The final 60,000 gmax peHet was resuspended in electrophoresis chamber buffer for free-flow electrophoretic separation of membranes as described (Sandelius et al., 1986). 

Energy-transducing processes in the cytoplasmic membrane of Rps. sphaeroides such as electron-transfer-linked proton translocation, and solute transport can be studied in whole cells or in isolated membrane vesicles wnich have retained their functional properties. In these latter systems the membrane-bound activities can be separated from activities in the cytoplasm. Two procedures have been described for the isolation of membrane vesicles. Mechanical disruption of the cells by sonication. French-press treatment or grinding with aluminium yields so-called chromatophores which are mainly derived from the intracytoplasmic membranes (Oelze, Drews, 1972). 

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