One‐ and two‐dimensional electrophoresis in micro‐slab gels

Poehling, Hans‐Michael; Neurhoff, Volker

doi:10.1002/elps.1150010205

Cited by 76 publications

(22 citation statements)

References 32 publications

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“…After resuspending in homogenization buffer (50 mM potassium phosphate, 50 mM potassium chloride, 10% (v/v) glycerol, 10 mM mercaptoethanol, 1 mM phenylmethyl sulfonyl fluoride, 0.5 mM aluminium fluoride, protease inhibitor cocktail [P 9599, Sigma]), the homogenate was filtered through Miracloth and subsequently centrifuged at 500g (20 min), 10,000g (20 min), 40,000g (30 min), and 100,000g (100 min). An aliquot of the 100,000g supernatant was loaded onto a nondenaturing 2%-19% acrylamide gradient micro gel (Poehling and Neuhoff 1980).…”

Section: Cell Fractionation and Nondenaturing Gradient Micro Gelsmentioning

confidence: 99%

The Arabidopsis PILZ group genes encode tubulin-folding cofactor orthologs required for cell division but not cell growth

Steinborn¹,

Maulbetsch²,

Priester³

et al. 2002

Genes Dev.

163

130

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Plant microtubules are organized into specific cell cycle-dependent arrays that have been implicated in diverse cellular processes, including cell division and organized cell expansion. Mutations in four Arabidopsis genes collectively called the PILZ group result in lethal embryos that consist of one or a few grossly enlarged cells. The mutant embryos lack microtubules but not actin filaments. Whereas the cytokinesis-specific syntaxin KNOLLE is not localized properly, trafficking of the putative auxin efflux carrier PIN1 to the plasma membrane is normal. The four PILZ group genes were isolated by map-based cloning and are shown to encode orthologs of mammalian tubulin-folding cofactors (TFCs) C, D, and E, and associated small G-protein Arl2 that mediate the formation of ␣/␤-tubulin heterodimers in vitro. The TFC C ortholog, PORCINO, was detected in cytosolic protein complexes and did not colocalize with microtubules. Another gene with a related, although weaker, embryo-lethal phenotype, KIESEL, was shown to encode a TFC A ortholog. Our genetic ablation of microtubules shows their requirement in cell division and vesicle trafficking during cytokinesis, whereas cell growth is mediated by microtubule-independent vesicle trafficking to the plasma membrane during interphase. The microtubule cytoskeleton plays important roles in both nondividing and dividing cells of eukaryotes, assisting in vesicle trafficking and mediating proper segregation of daughter chromosomes to opposite poles during mitosis (for review, see Cole and Lippincott-Schwartz 1995;Straight and Field 2000). In fission yeast, microtubules have also been implicated in maintaining cell shape and polarity (Sawin and Nurse 1998). Higher-plant cells form their own specific microtubule arrays, such as cortical hoops of interphase microtubules that organize cell elongation (Whittington et al. 2001), and two arrays related to the plant-specific mode of cell division (for review, see Lloyd and Hussey 2001). The preprophase band forms transiently at the onset of mitosis and marks the cortical division site (for review, see Smith 2001). The phragmoplast forms at the center of the division plane in late anaphase and guides the delivery of Golgiderived vesicles. Their fusion results in the formation and lateral expansion of the cell plate that matures into a cell wall and flanking plasma membranes (for review, see Staehelin and Hepler 1996). Both preprophase band and phragmoplast also contain actin filaments; however, their specific role in plant cell division is not understood at present (Smith 2001).Microtubules are polymerized from ␣/␤-tubulin heterodimers (for review, see Nogales 2000). Newly synthesized ␣-and ␤-tubulin polypeptides undergo a sequence of folding steps catalyzed by chaperones. Initially, the tubulins are associated with the hexameric prefoldin complex that passes them on to the cytosolic chaperonin complex (Geissler et al. 1998;Vainberg et al. 1998;Hansen et al. 1999; for review, see Leroux and Hartl 2000;Llorca et al. 2000). Whereas fully func...

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Section: Cell Fractionation and Nondenaturing Gradient Micro Gelsmentioning

confidence: 99%

The Arabidopsis PILZ group genes encode tubulin-folding cofactor orthologs required for cell division but not cell growth

Steinborn¹,

Maulbetsch²,

Priester³

et al. 2002

Genes Dev.

163

130

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“…Electrophoretical separation of proteins was performed in a 7.5% polyacrylamide gel by using the microgel system described by Poehling and Neuhoff (1980). Denaturating extraction for electrophoresis was carried out by mixing 10 mg of dried needle powder and 100 mg of Polyclar AT (Serva, Heidelberg, Germany) with 1 mL of boiling Laemmli (1970) buffer, followed by heating (95"C, 10 min) and centrifugation (12,00Og,8 min,4°C).…”

Section: Partia1 Enzyme Purification Sds-pace and Western Blottingmentioning

confidence: 99%

Coarse and Fine Control and Annual Changes of Sucrose-Phosphate Synthase in Norway Spruce Needles

1996

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The control of Suc synthesis constitutes an important factor in the regulation of Suc export, photosynthesis, and starch formation. One of the key enzymes in the control of Suc synthesis is SPS, and its activity can be regulated via metabolic fine control, coarse control by covalent modification, and by the amount of enzyme protein. Metabolic fine control is exerted by the allosteric effectors Glc-6-P and Pi. Glc-6-P, an intermediate of Suc synthesis, activates, whereas Pi inhibits SPS activity in most tissues (Doehlert and Huber, 1983; Huber et al., 198910;Reimholz et al., 1994). Some species exhibit a light-dependent covalent modification of SPS. Accordingly, the enzymes can be classified into three different types (for review, see Huber and Huber, 1992). Organisms such as maize and other monocotyledons show a light-induced increase in V , , , under nonlimiting substrate conditions. In other plants (e.g. spinach), V,,, is not influenced by a dark + light transition; in contrast, an increase in SPS activity in the presence of limiting substrate * The work was supported by grants from the Projekt Europaisches Forschungszentrum fiir Massnahmen zur Luftreinhaltung and the Deutsche Forschungsgemeinschaft (Graduiertenkolleg: Organismische Interaktionen in Waldokosystemen).

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“…One-dimensional discontinuous SDS-microPAGE was performed as described by Poehling and Neuhoff (21), except that gel chambers were sealed with plastic (Plasti-Dip, St. Paul, MN). Gels were 25 mm long, 25 mm wide, and 0.5 mm thick.…”

Section: Microelectrophoresismentioning

confidence: 99%

Electrophoretic Assay for Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase in Guard Cells and Other Leaf Cells of Vicia faba L.

Tarczynski

Outlaw²,

Arold³

et al. 1989

Plant Physiol.

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The ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) contents of guard cells and other cells of Vkia faba L. leaflet were determined. To prevent proteolysis, proteins of frozen protoplast preparations or of cells excised from freeze-dried leaf were extracted directly in a sodium-dodecyl-sulfate-containing solution, which was heated immediately after sample addition. Tissue Preparation and Protein ExtractionTwo different procedures, each utilizing plants from three different growth lots, were used:1. Guard-cell protoplast preparations were made by differential digestion essentially as described as method 3 by Gotow et al. (3), except pepstatin A was not included in the digestion mixture, and epidermal peels were brushed to lower contamination. The protoplasts that were released after 180 min were further purified by differential sieving through a cascade of nylon meshes (210, 70, and 20 ,um)

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One‐ and two‐dimensional electrophoresis in micro‐slab gels

Cited by 76 publications

References 32 publications

The Arabidopsis PILZ group genes encode tubulin-folding cofactor orthologs required for cell division but not cell growth

The Arabidopsis PILZ group genes encode tubulin-folding cofactor orthologs required for cell division but not cell growth

Coarse and Fine Control and Annual Changes of Sucrose-Phosphate Synthase in Norway Spruce Needles

Electrophoretic Assay for Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase in Guard Cells and Other Leaf Cells of Vicia faba L.

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