Membrane protein extract

Figure 4. Purification of PemB from E. coli K38 pGPl-2/pPME6-5 cells. Proteins were separated by urea-SDS-PAGE. Lane 1, induced cell lysate lane 2, soluble protein fraction from induced cells lane 3, membrane fraction from non-induced cells lane 4, membrane fraction from induced cells lane 5, membrane proteins not extracted by Triton X-100 lane 6, membrane proteins extracted by Triton X-100 lane 7, PemB purified by preparative electrophoresis. The molecular weight standard positions are indicated.

In one study by Hood et al., 282 of 1153 identified proteins were identified by at least 2 unique tryptic peptides from FFPE prostate cancer (PCa) tissue.9 According to the gene ontology classification of the proteins identified, -65% of proteins were predicted to be intracellular proteins, while -50% of the total human proteome is predicted to be located in the intracellular compartment. Additionally, 20% of the proteins identified in the PCa tissue were classified as membrane proteins, which is significantly less than the predicted 40% for the human proteome. This relative disparity is not unexpected, considering the Liquid Tissue sample preparation kit lacks specific protocols for membrane protein extraction. The Liquid Tissue method has also been used for proteomics studies of a variety of FFPE tissue samples, including pancreatic tumors,28 squamous cell carcinoma,4 and oral human papillomavirus lesions.27... 

Incubate with the specific antigen (50 pL per well of a 0.1-1 pg/ mL solution in PBST) for 2 h at 37°C, or overnight at 4-6 C, then wash with PBST. For antigens that are not readily soluble, e.g., membrane proteins extracted in nonionic detergents, the extract in 0.5-1 % Triton X-100 or Nonidet-P40 can be used after suitable dilution with PBSA containing 0.5% of the detergent All subsequent procedures should be carried out... 

Figure 6. Time depenc3ence of resistance ( ) and capacitance (O) of bilayer lipid membranes on asymnetric addition of Triton X-100 membrane protein extracts at 27 °C. (a) Bathing solution O.lM...

Jabbour RE, Wade MM, Deshpande SV, et al. Identification of Yersinia pestis and Escherichia coli strains by whole cell and outer membrane protein extracts with mass spectrometry-based proteomics. J Proteome Res. 2010b 9 3647-55. doi 10.1021/prl00402y. 

One area of rapidly expanding interest is the use of reverse micellar systems of sugar-based surfactants in the extraction of proteins and other sensitive materials. The use of hydrophilic, nonionic, sugar-based surfactants for membrane protein extraction is well known to be effective due to the mild, nondenaturing properties of these surfactants when compared with ionic surfactants or polyoxyethylene derivatives. For the same reasons, protein extraction into reverse micellar systems is now becoming a popular medium for such applications. Alkyl sorbitan esters and ethoxylated sorbitan esters, such as Tween 85 [107] and Span 60 [108], have been used successfully to form reverse micellar systems for protein extraction. Blends of Tween and Span have also been found to be effective for this purpose [109]. More recently, commercially available sucrose fatty acid esters have been shown to form biocompatible reverse micellar systems into which cytochrome c is effectively extracted [110]. 

Scientists initially approached structure-function relationships in proteins by separating them into classes based upon properties such as solubility, shape, or the presence of nonprotein groups. For example, the proteins that can be extracted from cells using solutions at physiologic pH and ionic strength are classified as soluble. Extraction of integral membrane proteins requires dissolution of the membrane with detergents. 

The PemB cellular localisation was determined both in E. chrysanthenu and in an E. coli recombinant strain by Western blot of the cell fractions with a PemB-antiserum. No PemB was detected in the culture supernatant and only trace amounts were found in the soluble cell fractions - periplasm and cytoplasm (Figure 2). PemB was found mostly in the total membrane fraction from which it could be completely extracted by Triton X-100/Mg2+ and partially extracted by Sarkosyl (Figure 2). This behaviour is typical of inner membrane proteins, but since some exceptions have been noticed it does not positively indicate the PemB localisation (15). We performed cell membrane fractionation in sucrose density gradient centrifugation both by sedimentation and flotation, using several markers of inner and outer membrane vesicles. PemB was found in the outer membrane vesicles (data not shown). 

Stability of several enzymes like proteases from thermophilic micro-organisms can be increased in aqueous-organic biphasic systems. Owusu and Cowan [67] observed a strong positive correlation between bacterial growth temperature, the thermostability of free protein extracts, and enzyme stability in aqueous-organic biphasic systems (Table 1). Enzymes, like other cell components (membranes, DNA, (RNA ribosomes), are adapted to withstand the environmental conditions under which the organism demonstrates optimal growth. 

Penetration enhancers are low molecular weight compounds that can increase the absorption of poorly absorbed hydrophilic drugs such as peptides and proteins from the nasal, buccal, oral, rectal, and vaginal routes of administration [186], Chelators, bile salts, surfactants, and fatty acids are some examples of penetration enhancers that have been widely tested [186], The precise mechanisms by which these enhancers increase drug penetration are largely unknown. Bile salts, for instance, have been shown to increase the transport of lipophilic cholesterol [187] as well as the pore size of the epithelium [188], indicating enhancement in both transcellular and paracellular transport. Bile salts are known to break down mucus [189], form micelles [190], extract membrane proteins [191], and chelate ions [192], While breakdown of mucus, formation of micelles, and lipid extraction may have contributed predominantly to the bile salt-induced enhancement of transcellular transport, chelation of ions possibly accounts for their effect on the paracellular pathway. In addition to their lack of specificity in enhancing mem-... 

Fig. 1.4 Protein blot analysis of C5-1 assembly in agroinfiltrated alfalfa leaves. Total leaf soluble proteins, extracted 4 days after infiltration were separated by SDS-PAGE under non-reducing conditions and blotted onto a PVDF membrane. Polyclonal antimouse IgGs were used for detection. Purified C5-1 was mixed with total soluble proteins from control infiltrated alfalfa leaves and loaded as a standard.

To date, the lipids so far used have been mainly extracts from natural sources such as EPC and archaeal lipids[17,18] The chemical stability of EPC, however, is not sufficient and the membrane permeability to H1 is sometimes too high for quantitative analyses of membrane protein functions. Though archaeal lipids display many preferable features for... 

Protegrin derivatives, 18 260 Protegrins, 18 260-261 properties of, 18 261 Protein. See also Proteins extraction of, 26 474 in cereal grains, 26 275-276 Proteinaceous materials, as membrane foulants, 21 664 Protein adsorption, 12 136-137 Protein affinity libraries, 12 516-517 Protein-based chiral phases, 6 89-90 Protein-based microarrays, 16 382 Protein biosynthesis, 20 450... 

A number of studies have taken advantage of the fact that membrane proteins contain one or more tryptophans, the fluorescence of which can be used to determine the conformation of the protein or its position in the membrane.(88 91) Of course, the information is limited by the number of tryptophans and the fact that a tryptophan may not be positioned in the region of the protein of interest. While a single tryptophan often simplifies the situation, most often there are a number in the protein so that it is difficult to extract useful information. 

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. 

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