16-BAC/SDS–PAGE: A Two-Dimensional Gel Electrophoresis System Suitable for the Separation of Integral Membrane Proteins

Hartinger, Joachim; Stenius, Katinka; Högemann, Dagmar; Jahn, Reinhard

doi:10.1006/abio.1996.0339

Cited by 189 publications

(146 citation statements)

References 7 publications

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“…Approximately 500 g of synaptic vesicle proteins were separated by 16BAC/SDS-PAGE (16). After Coomassie Blue staining, all visible spots were excised, cut into ϳ1-mm 2 pieces, and subjected to in-gel trypsinization (17).…”

Section: Methodsmentioning

confidence: 99%

Identification of SNAP-47, a Novel Qbc-SNARE with Ubiquitous Expression

Holt

Varoqueaux

Wiederhold

et al. 2006

Journal of Biological Chemistry

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123

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The SNARE proteins are essential components of the intracellular fusion machinery. It is thought that they form a tight four-helix complex between membranes, in effect initiating fusion. Most SNAREs contain a single coiled-coil region, referred to as the SNARE motif, directly adjacent to a single transmembrane domain. The neuronal SNARE SNAP-25 defines a subfamily of SNARE proteins with two SNARE helices connected by a longer linker, comprising also the proteins SNAP-23 and SNAP-29. We now report the initial characterization of a novel vertebrate homologue termed SNAP-47. Northern blot and immunoblot analysis revealed ubiquitous tissue distribution, with particularly high levels in nervous tissue. In neurons, SNAP-47 shows a widespread distribution on intracellular membranes and is also enriched in synaptic vesicle fractions. In vitro, SNAP-47 substituted for SNAP-25 in SNARE complex formation with the neuronal SNAREs syntaxin 1a and synaptobrevin 2, and it also substituted for SNAP-25 in proteoliposome fusion. However, neither complex assembly nor fusion was as efficient as with SNAP-25.Eukaryotic cells are compartmentalized into membrane-enclosed organelles that communicate with each other by membrane traffic. Each trafficking step involves three basic steps: the formation of an organelle by budding from a precursor, transport of the vesicle to its destination along cytoskeletal tracks, and finally docking and fusion of the organelle with its target membrane (reviewed in Refs. 1 and 2). Docking and fusion are mediated by the ordered assembly of multimolecular protein complexes that form upon organelle contact and that are disassembled after fusion is completed. Tethering and docking require large arrays of proteins, many of which are specific for a given transport step. Fusion itself, however, is catalyzed by a set of conserved proteins termed soluble N-ethylmaleimidesensitive factor attachment protein receptors (SNAREs) 4 (3, 4).SNARE proteins comprise a superfamily of mostly membranebound proteins with at least 24 members in yeast and 35 members in mammals (5, 6). The characteristic feature of all SNAREs is the presence of a stretch of ϳ60 amino acids arranged in heptad repeats that is referred to as a SNARE motif. In most SNAREs, SNARE motifs are positioned adjacent to a transmembrane domain at the C terminus. Exceptions include SNAREs that lack a transmembrane domain and contain two SNARE motifs, instead of one, which are connected by a flexible linker, such as mammalian SNAP-25, SNAP-23, and SNAP-29/GS32. SNAP-25 and SNAP-23 contain multiple palmitate residues attached to cysteine residues in the linker region (7, 8), whereas SNAP-29 lacks a membrane anchor altogether (9, 10).SNARE proteins undergo an assembly-disassembly cycle that is mediated by the SNARE motifs. Each of the membranes destined to fuse contains sets of SNAREs that are complementary to each other. Before membrane merger, the SNAREs are thought to connect with each other in a "trans-configuration," forming an ␣-helical bundle of four SNARE...

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Section: Methodsmentioning

confidence: 99%

Identification of SNAP-47, a Novel Qbc-SNARE with Ubiquitous Expression

Holt

Varoqueaux

Wiederhold

et al. 2006

Journal of Biological Chemistry

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123

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“…To deplete soluble and membrane-associated proteins, this pellet was additionally subjected to six consecutive washing/centrifugation cycles (2 ϫ 50 mM Tris, 150 mM NaCl, 1 ϫ 1 M KCl, 1 ϫ 0.1 M Na 2 CO 3 , 2 ϫ 2 mM EGTA). The final pellet was resuspended in 100 l of 16-BAC sample buffer (22.5% urea, 5% 16-BAC, 5% glycerol, 37.5 mM DTT, 0.025% Pyronin Y) and separated on 7.5% acrylamide 16-BAC gels (20 ϫ 20 cm) as described previously (Hartinger et al, 1996). After the first dimension, gels were stained with Coomassie brilliant blue (CBB) R250.…”

Section: Animals Plpmentioning

confidence: 99%

Proteolipid Protein Is Required for Transport of Sirtuin 2 into CNS Myelin

Werner

Kuhlmann

Shen³

et al. 2007

J. Neurosci.

228

267

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Mice lacking the expression of proteolipid protein (PLP)/DM20 in oligodendrocytes provide a genuine model for spastic paraplegia (SPG-2). Their axons are well myelinated but exhibit impaired axonal transport and progressive degeneration, which is difficult to attribute to the absence of a single myelin protein. We hypothesized that secondary molecular changes in PLP null myelin contribute to the loss of PLP/DM20-dependent neuroprotection and provide more insight into glia-axonal interactions in this disease model. By gel-based proteome analysis, we identified Ͼ160 proteins in purified myelin membranes, which allowed us to systematically monitor the CNS myelin proteome of adult PLP null mice, before the onset of disease. We identified three proteins of the septin family to be reduced in abundance, but the nicotinamide adenine dinucleotide (NAD ϩ )-dependent deacetylase sirtuin 2 (SIRT2) was virtually absent. SIRT2 is expressed throughout the oligodendrocyte lineage, and immunoelectron microscopy revealed its association with myelin. Loss of SIRT2 in PLP null was posttranscriptional, suggesting that PLP/DM20 is required for its transport into the myelin compartment. Because normal SIRT2 activity is controlled by the NAD ϩ /NADH ratio, its function may be coupled to the axo-glial metabolism and the long-term support of axons by oligodendrocytes.

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“…Recently 16-BAC/ SDS-PAGE has gained broad application for the separation of membrane and hydrophobic proteins (17,18). This technique is based on the consecutive separation of proteins by means of electrophoretic mobility using two different detergents, the cationic 16-BAC in the first and the anionic SDS in the second dimension (19). In comparison to 2D-PAGE the resolution is reduced but still much better than in one-dimensional gel systems.…”

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confidence: 99%

The Human Platelet Membrane Proteome Reveals Several New Potential Membrane Proteins

Moebius¹,

Zahedi²,

Lewandrowski³

et al. 2005

Molecular & Cellular Proteomics

152

134

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We present the first focused proteome study on human platelet membranes. Due to the removal of highly abundant cytoskeletal proteins a wide spectrum of known platelet membrane proteins and several new and hypothetical proteins were accessible. In contrast to other proteome studies we focused on prefractionation and purification of membranes from human platelets according to published protocols to reduce sample complexity and enrich interesting membrane proteins. Subsequently protein separation by common one-dimensional SDS-PAGE as well as the combined benzyldimethyl-n-hexadecylammonium chloride/SDS separation technique was performed prior to mass spectrometry analysis by nano-LC-ESI-MS/MS. We demonstrate that the application of both separation systems in parallel is required for maximization of protein tagging out of a complex sample.

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16-BAC/SDS–PAGE: A Two-Dimensional Gel Electrophoresis System Suitable for the Separation of Integral Membrane Proteins

Cited by 189 publications

References 7 publications

Identification of SNAP-47, a Novel Qbc-SNARE with Ubiquitous Expression

Identification of SNAP-47, a Novel Qbc-SNARE with Ubiquitous Expression

Proteolipid Protein Is Required for Transport of Sirtuin 2 into CNS Myelin

The Human Platelet Membrane Proteome Reveals Several New Potential Membrane Proteins

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