Membrane solubilization

Some cell lines, such as HEK293, may detach during the permeabilization step due to a strong Ca2+ dependence for attachment. While it is critical that cells are permeabilized as a monolayer, detachment does not seem to hinder cytosolic ribosome release, as they tend to detach as a (partial) monolayer. Following the permeabilization step, cells can simply be separated from the soluble cytosol phase by centrifugation at 750-1000 Xg for 5 min. Transfer the supernatant (cytosol) to a new tube. Remove any remaining cells attached to the flask via the wash buffer, combine with the cell pellet, and recover by centrifugation. Proceed with the membrane solubilization step. 

Anionic and cationic surfactants are more effective in membrane solubilization than nonionic surfactants. 

Thrbidity is a spectroscopic technique determining the optical density of colloidal particles. A wavelength between 350 and 500 nm is the first choice for such studies [56], Thrbidity measurements can offer important information on the kinetics of membrane-surfactant interactions since membrane solubilization changes reflect changes to the optical density of the dispersion [57-60], It is also widely used as a technique to investigate liposome aggregation and fusion [61]. However the exact particle size of liposomes cannot be determined using turbidity techniques. 

Goni, F. M., and Alonso, A. (2000), Spectroscopic techniques in the study of membrane solubilization, reconstitution and permeabilization by detergents, Biochim. Biophys. Acta, 1508, 51-68. 

The polypeptides of the light-harvesting complex are most easily resolved by polyacrylamide gel electrophoresis of thylakoid membranes solubilized with sodium or lithium dodecyl sulphate. This produces the green chlorophyll-protein band, CP II, which on staining for protein reveals up to four polypeptides of 24-27 kDa [2], each of which is believed to bind Chi a and Chi b. In most plants two polypeptides predominate, but other minor polypeptides may be resolved [2]. 

Allan, Auger and Crumpton used Con A-Sepharose to isolate glycoprotein cell surface receptors for concanavalin A from pig lymphocyte membranes solubilized with sodium deoxycholate [134]. 

Disrupt membrane Solubilize protein with detergents... 

The early work on the interactions of synthetic surfactants and fatty acid anions with proteins was reviewed by Putnam [9] in 1948 and the following 20 years by Steinhardt and Reynolds [10]. The expansion of the field has led more recently to reviews and monograph chapters dealing with specific areas of protein-surfactant interactions, such as the solubilization of membranes by detergents [11] and the use of surfactants in membrane solubilization and reconstitution of membrane proteins [12], as well as more general reviews [13,14,15],... 

Other work has been on membrane topology, and antibody studies indicate that P450 8A1 is mainly exposed on the cytoplasmic site of the endoplasmic reticulum with a single transmembrane anchor . The (imstable) substrate, prostaglandin H2, is produced in the lumen and apparently passes through the membrane to reach P450 8A1. Antibodies raised to the peptides of the putative substrate channel (66-75 and 95-116) interact only after membrane solubilization, implying that the substrate-access channel is very near the membrane . 

Essentially similar results have been obtained by Eylar and co-workers " on the biosynthesis of bovine submaxillary mucin. The enzyme is firmly bound to a membrane solubilization by Triton X-100 results in a large increase in enzymic activity." The enzyme was purified by gel filtration, and its properties were studied. Among various substances studied, the only one (besides carbohydrate-free mucins) that was active as the acceptor of 2-acetamido-2-deoxy-D-galactose was a basic protein, called encephalitogen, isolated from bovine, spinal-cord myelin. ... 

Thomas J L, Barton S W, Tirrell D A (1994). Membrane solubilization by a hydro-phobic polyelectrolyte surface activity and membrane binding. Biophys. J. 67 1101-1106. 

Ghoroghchian PP, Frail PR, Susumu K, Park TH, WU SP, Uyeda HT, Hammer DA, Therien MJ (2005) Broad spectral domain fluorescence wavelength modification of visible and near-infrared emissive polymersomes. J Am Chem Soc 127 15388-15390 Ghoroghchian PP, Jin JJ, Brannan AK, Frail PR, Bates FS, Therien MJ, Hammer DA (2006) Quantitative membrane loading of polymer vesicles. Soft Matter 2 973-980 Pata V, Ahmed F, Discher DD, Dan N (2004) Membrane solubilization by detergent resistance conferred by thickness. Langmuir 20(10) 3888-3893... 

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. 

The chemical shifts of various phospholipids in organic solvent and Triton X-100 micelles are shown in Tables 9.13 and 9.14. A detailed discussion on P-NMR of phospholipids in micelles, including applications to membrane solubilization by detergents and phospholipase activity may be found in the recent review of Dennis and Pliickthun (1984). 

Similar calculations have been Initiated to ascertain the relative midpoints of the two bacterlochlorophylls (Bh and Bl) and the two bacteriopheophytins (Hl and Hm)- The assumptions used were the same as those outlined above for the cytochrome calculations, except that glutamic acid L104 is uncharged. It has been found that this residue can be replaced with non-polar amino acids without loss of function (30). These calculations suggest that the L side is only very slightly easier to reduce than the M branch, the differences between Bl and Bm and between Hl and Hm are less than 60 meV. Similar results were found when the protein was assumed be surrounded by water or by a low dieletric, such as a membrane. RC function with little change in the native membrane, solubilized with detergent or even suspended in hexane (31). 

Fig.3-SDS-PAGE of the Chlorophyll-protein complexes of membranes solubilized at different temperatures.

In the sample preparation step, the nucleic acid content from the sample is extracted, concentrated, and purified to meet the quality required by the downstream amplification process. Sample preparation of nucleic acid targeting technologies involves several processes. In most cases, lysis is an initially required process to break open intact structures of targeted species (e.g., cell wall, membrane, and capsules) and to release nucleic acids. Additional purification or extraction of the released nucleic acids from the lysate may be conducted to minimize inhibitory effects on downstream amplification. Following the lysis step, the chemicals introduced (e.g., membrane-solubilizing ionic detergents or proteinases), cell debris, and other interferents that come with samples are removed. For diluted specimens (i.e., urine), nucleic acid concentration may be required in order to reduce the limit of detection of amplification and detection assays. 

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