Assessment of four different detergents used to extract membrane proteins from Xylella fastidiosa by two-dimensional electrophoresis

Ciero, Luciana Di; Bellato, Cláudia de M.; Meinhardt, Lyndel W.; Ferrari, Fernanda; Castellari, Rafael Ramos; Marangoni, Sérgio; Novello, José Camillo

doi:10.1590/s1517-83822004000200018

Cited by 5 publications

(4 citation statements)

References 29 publications

(12 reference statements)

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“…Figure 2A shows that the total amount of proteins solubilized from erythrocyte ghosts by ASB-14 and ASB-16 is higher than that obtained with CHAPS or Triton X-100, even at the highest concentrations of detergents tested. This result agrees well with the two dimensional electrophoresis data in the literature that show an improvement in the resolution of many integral membrane proteins (11-16) with ASB-14 and ASB-16 compared with classic detergents such as Triton and CHAPS (22,23). We have tested both detergents in the sample buffer used for twodimensional gel electrophoresis of red blood cells, and the results indicate that different proteins were solubilized by using these detergents, and therefore ASB detergents are useful to achieve a more detailed assignment of the proteins (24).…”

Section: Resultssupporting

confidence: 91%

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Solubilization of human erythrocyte membranes by ASB detergents

Domingues

Malheiros

Paula

2008

Braz J Med Biol Res

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Understanding the membrane solubilization process and finding effective solubilizing agents are crucial challenges in biochemical research. Here we report results on the interaction of the novel linear alkylamido propyl dimethyl amino propanosulfonate detergents, ASB-14 and ASB-16, with human erythrocyte membranes. An estimation of the critical micelle concentration of these zwitterionic detergents (ASB-14 = 100 µM and ASB-16 = 10 µM) was obtained using electron paramagnetic resonance. The amount of proteins and cholesterol solubilized from erythrocytes by these detergents was then determined. The hemolytic activities of the ASB detergents were assayed and the detergent/lipid molar ratios for the onset of hemolysis (R e sat ) and total lysis (R e sol ) were calculated, allowing the determination of the membrane binding constants (K b ). ASB-14 presented lower membrane affinity (K b = 7050 M -1 ) than ASB-16 (K b = 15610 M -1 ). The amount of proteins and cholesterol solubilized by both ASB detergents was higher while R e sat values (0.22 and 0.08 detergent/lipid for ASB-14 and ASB-16, respectively) were smaller than those observed with the classic detergents CHAPS and Triton X-100. These results reveal that, besides their well-known use as membrane protein solubilizers to enhance the resolution of two dimensional electrophoresis/ mass spectrometry, ASB-14 and ASB-16 are strong hemolytic agents. We propose that the physicochemical properties of ASB detergents determine their membrane disruption efficiency and can help to explain the improvement in the solubilization of membrane proteins, as reported in the literature.

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Section: Resultssupporting

confidence: 91%

“…Our results suggest that the relationship between membrane disruption efficiency (Table 2) and the physicochemical properties of ASB detergents (CMC, HLB) can explain the improvement in the solubilization of membrane proteins, as described in proteomic research papers (11)(12)(13)(14)(15)(16)22,23).…”

Section: Hemolytic Experimentssupporting

confidence: 64%

Solubilization of human erythrocyte membranes by ASB detergents

Domingues

Malheiros

Paula

2008

Braz J Med Biol Res

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“…This was in agreement with previous observations for the solubilization of membrane proteins. 23,24 Surprisingly, CHAPS, which was the most commonly used detergent, had the worst solubilization power for skeleton proteins. This was probably caused by the high molecular weight and low solubility of these proteins.…”

Section: Resultsmentioning

confidence: 99%

Proteomic Analysis of a Membrane Skeleton Fraction from Human Liver

Liu

et al. 2007

J. Proteome Res.

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The cytoskeleton networks around liver cell cortex can resist Triton extraction and co-pellet with their tightly associated integral membrane proteins, forming assemblies called "membrane skeletons". Despite their important roles in determining cell shape and in signal transduction pathways, the membrane skeletons of human liver cells are uncharacterized to a great extent. In the present work, we prepared a membrane skeleton fraction by Triton extraction of human liver plasma membranes and then separated its protein components by 2-D gels. We optimized the detergent used for protein solubilization and found that 2% ASB-14 allowed the best recovery of membrane skeleton proteins. By analyzing the protein spots with MALDI-TOF and MALDI-TOF-TOF MS, we identified 104 nonredundant proteins, wherein 38 were cytoskeletal proteins that were further classified into several groups, including proteins in fodrin-based meshworks, adhesion proteins (proteins involved in adherens junctions, focal adhesions, desmosomes, hemidesmosomes and tight junctions), proteins that regulate F-actin dynamics, motor proteins, and some other cytoskeletal proteins. To the best of our knowledge, this is one of the largest data sets of membrane skeleton proteins to date. All the results suggested that the liver cells had complex actin- and cytokeratin-based membrane skeletons. This work provided a representative 2-DE map of membrane skeletons from human normal liver, for the purpose of helping to elucidate the composition and function of the membrane skeletons.

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“…Combined to a chaotrope, ASB-14 improves the solubilization of hydrophobic proteins [20]. It has been successfully used on rat brain tissues to solubilize both cytosolic and membrane proteins [21] and has increased the proportion of membrane proteins extracted from Xyllela fastidiosa [22]. An efficient protein extraction from human cardiac tissues was obtained with an initial homogenization in a buffer containing ASB-14 followed by focusing in a buffer containing CHAPS [23].…”

Section: Protocol Comparisonmentioning

confidence: 99%

Optimization of protein extraction and solubilization for mature grape berry clusters

2006

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Protein extraction from grape berries has been challenging, particularly in mature berries, which can have sugar concentrations as high as 26%. Grape skins and seeds contain large amounts of polyphenols, which can also interfere with efficient protein extraction. In plants, two extraction protocols, TCA/acetone-based and phenol-based methods, have been mainly used to extract proteins from different organs or tissues on many species. However, few results have been reported for grape berry clusters. We wanted to determine which of these protocols was optimal for berry clusters in order to achieve both efficient protein extraction and high spot resolution on 2-D gels. Four protocols, derived from either TCA/acetone or phenol procedures, were tested on mature Cabernet Sauvignon whole berry clusters. The phenol-based protocols were superior to the TCA/acetone methods, showing larger protein yields and greater spot resolution on 2-D gels. One method was clearly superior to the rest, a phenol-based extraction method combined with resuspension in the presence of both urea and thiourea as chaotropes. A total of 81 spots were excised and identified following MALDI-TOF/TOF MS analyses. Their identification helped further characterize the specificity of each extraction procedure.

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Assessment of four different detergents used to extract membrane proteins from Xylella fastidiosa by two-dimensional electrophoresis

Cited by 5 publications

References 29 publications

Solubilization of human erythrocyte membranes by ASB detergents

Solubilization of human erythrocyte membranes by ASB detergents

Proteomic Analysis of a Membrane Skeleton Fraction from Human Liver

Optimization of protein extraction and solubilization for mature grape berry clusters

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