Solubilized membrane extracts

Penetration of Solubilized Membrane Extracts into Monolayers... 

There are two approaches to isolating a membrane-associated protein. In one method, the relevant membrane fraction can first be prepared and then used to isolate the protein. Alternatively, whole tissue can be subjected to an extraction that solubilizes the membranes and releases the cytoplasmic contents as well. The former is much better in that purification is accomplished by isolating the membranes the specific activity of the solubilized membrane fraction will be much higher than in the second method. However, the process of purifying the membrane fraction may lead to substantial losses, and it may be difficult to scale up. If total recovery of the protein is more important than purity, a whole-tissue extract is likely to be more appropriate. Although this... 

Chloroform-methanol is used to extract lipid and lipoprotein material according to Folch et al. [147], with the addition of aqueous KC1 to isolate lipid and lipoproteins. Alternatively, if lipid material is not to be investigated, fats can be removed with acetone with little co-extraction of Se. The presence of fats is not desirable as lower aqueous extraction efficiencies are obtained and nondefatted samples can degrade HPLC performance. Extraction with phosphate buffer will remove cytosol-soluble Se, while extraction with phosphate buffer containing SDS will release and solubilize membrane-bound Se. Phenylmethane sulfonyl fluoride or a similar reagent needs to be added to buffers to retard proteolytic... 

Non-sulfur, purple, photosynthetic bacteria, Rho do spirillum rub-rum and Rhodopseudomonas spheroides172 also possess a PEP-de-pendent D-fructose phosphotransferase. Two protein fractions are required for D-fructose phosphorylation. In contrast to PEP-depend-ent, phosphotransferase systems isolated from other bacteria, the aforementioned two organisms have one active protein fraction tightly associated with the membrane fraction, while another in the crude extract is solubilized by extraction with water, and has a molecular weight of about 200,000. There is no evidence for the presence of a phosphate-carrier protein of low molecular weight like HPr.171,173 The... 

Triton X-100 extracts of erythrocyte membrane proteins can be incorporated into BLMs provided excess Triton X-100 is removed by gel filtration (Sephadex G50) to bring the free detergent level to approximately l g cm . By monitoring the electrical characteristics of the BLM on exposure to Triton X-100 solubilized protein extracts the penetration process can be followed. 

In membrane extraction of metals, the mass transport of solute from one phase to another occurs by diffusion. It is controlled by phase equilibrium and the resistances of boundary layers in two phases and the membrane material. Both types of materials are used for membrane extraction and stripping, hydrophilic and hydrophobic, and composite hydro-philic/hydrophobic barriers are also developed to avoid the membrane solubilization [122,123]. To enhance separation, the reactive liquids that induce chemical reaction with one of the separated species can be used. In membrane SX of metals, extracting agents, such as tri- -octylphosphine oxide (TOPO), di(2-ethylhexyl)phosphoric acid (D2EHPA), and n-octyl(phenyl)-A,A-diisobutylcarbamoylmethylphosphine oxide (CMPO), and commercial reagents like CYANEX 301, CYANEX 923, LIX622, and LIX622N are applied. 

Figure 3. Changes in the ratio of the two forms of D1 in response to decreasing light availability. An 8-liter culture of wild-type cells was illuminated at a constant PPFD of 600 pE-m"2-s l and the light at the interior of the culture was measured at time points diuring the development of the culture. Samples of cells were collected at each point and thylakoid membranes were extracted. Solubilized membrane proteins (80pg) from each sample were separated by LiDS polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and reacted with antisera directed against form I, form II or (combined) D1 as indicated. Each lane is marked with a number representing the light intensity, in pE-m -s l, inside the culture at the time point used to prepare the sample in that lane.

The advantage of these detergents is that solubilized membrane proteins are still biologically active after extraction. 

In Table 2, the values of n are presented for the intact membrane, solubilized membrane, reaggregated material and Triton X-100 solubilized reaggregated material from animals fed corn oil supplemented diet and fat-free diet, respectively. As it can be observed, the value of n is around 1.6 for all the enzyme preparations from the fat sufficient animals. For the preparations from the fat deficient animals instead it is in the order of 1.0 in the case of intact membrane and reaggregation membrane like material, and around 1.6 for the solubilized preparations. Similar results were obtained with the other solubilizing agents (Martinez de Melian et al., 1976). Further evidence on the role played by the membrane was obtained when the acetylcholinesterase band from the gel electrophoresis of solubilized acetylcholinesterase from fat sufficient animals was eluted, mixed with lipid extracted from red cell membranes of rat fed a fat-free diet, and diffused against buffer. A value of n=1.0, which corresponds to that of the intact membrane from the deficient... 

In contrast, the study of proteins present in minute amounts and/or of restricted localization may require selective protein extraction. Several procedures have been described including use of nuclei (Sormenberg et al., 1989) modified by Hope (Hope et al., 1994), sarcolemmal-enriched membrane fractions (Tuana et al., 1987), apoHpoprotein (Peitsch et aL, 1989), or microsome fractions (Kumar et al., 1985) for protein extractions. Numerous others exist and as a general rule, they rely first on the isolation of the cellular compartment of interest followed by the solubilization of the proteins contained in that cellular fraction. 

Solubilization of Membrane Proteins. A modification of the procedure of Hjelmeland et al. (30) was employed. A 300-g portion of liquid-nitrogen frozen, 6-day Nicotiana silvestris cultured cells was suspended in 200 ml of 50 mM N-(2-hydroxyethyl)-piperazine-N -3-propanesulfonic acid (EPPS-KOH) buffer, 1 mM dithiothreitol (DTT), and 0.1 mM EDTA extraction buffer with constant stirring until completely suspended (20 min). The slurry was centrifuged in a Sorvall SS 34 rotor at 9,000g for 20 min at 4°C. The supernatant was passed through miracloth (Calbiochem). An aliquot... 

Preparation of Glucan Synthase Fractions. Microsomal and plasma membranes were isolated by differential and density-gradient centrifugation. CHAPS-solubilized glucan synthase (CSGS) was prepared by the two-step procedure (4,9). In step 1, contaminating proteins were extracted with... 

Molloy MP, Herbert BR, Walsh BJ, et al. (1998) Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis 19, 837- 4. 

Extractant leakage from the pores of the polymeric membrane in SLM is due to osmotic flow of massive quantities of water through the membrane. Membrane stability decreases with increasing osmotic pressure gradient and depends upon composition of the SLM system. A high tendency to solubilize water, low extractant/aqueous interfacial tension, and high wettability of polymeric membrane leads to less stable SLMs. The following measures have been proposed for improvement of stability ... 

In many cases, enzymes of interest are associated with insoluble parts of the cell, such as mitochondria, endoplasmic reticulum or membranes, and an extraction procedure is necessary. Not all of these enzymes can survive solubilization in the absence of their normal cellular milieu. The extraction buffers should not only have the correct pH and ionic strength but also the appropriate stabilizers should be added (Table 5.17). 

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