A Simple, Versatile, Sensitive and Volume-Independent Method for Quantitative Protein Determination which is Independent of Other External Influences

Neuhoff, Volker; Philipp, Klaus; Zimmer, Hans-Georg; Mesecke, Senta

doi:10.1515/bchm2.1979.360.2.1657

Cited by 234 publications

(119 citation statements)

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“…Protein concentrations in the samples were determined according to published methods. 41 Total cellular extracts were separated by one-dimensional SDS-PAGE using 7.5% polyacrylamide gels followed by Western blot analysis with n847 and the 17024 anti-tau antibody as described above. For FIHC, cells were cultured for 2 days on poly-L-lysine-coated glass cover-slips (2 ϫ 10 4 cells per 35-mm dish) in DMEM/0.5%FBS, and treated with 0.5 mmol/L peroxynitrite.…”

Section: Cell Culturementioning

confidence: 99%

Nitration of Tau Protein Is Linked to Neurodegeneration in Tauopathies

Horiguchi

Uryu

Giasson

et al. 2003

The American Journal of Pathology

182

118

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Oxidative and nitrative injury is implicated in the pathogenesis of Alzheimer's disease (AD) and Down syndrome (DS), but no direct evidence links this type of injury to the formation of neurofibrillary tau lesions. To address this, we generated a monoclonal antibody (mAb), n847, which recognizes nitrated tau and ␣-synuclein. n847 detected nitrated tau in the insoluble fraction of AD, corticobasal degeneration (CBD), and Pick's disease (PiD) brains by Western blots. Immunohistochemistry (IHC) showed that n847 labeled neurons in the hippocampus and neocortex of AD and DS brains. Double-label immunofluorescence with n847 and an anti-tau antibody revealed partial co-localization of tau and n847 positive tangles, while n847 immmunofluorescence and Thioflavin-S double-staining showed that a subset of n847-labeled neurons were Thioflavin-S-positive. In addition, immuno-electron microscopy revealed that taupositive filaments in tangle-bearing neurons were also labeled by n847 and IHC of other tauopathies showed that some of glial and neuronal tau pathologies in CBD, progressive supranuclear palsy, PiD, and frontotemporal dementia with parkinsonism linked to chromosome 17 also were n847-positive. Finally, nitrated and Thioflavin-S-positive tau aggregates were generated in a oligodendrocytic cell line after treatment with peroxynitrite. Taken together, these findings imply that nitrative injury is directly linked to the formation of filamentous tau inclusions. (Am J

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Section: Cell Culturementioning

confidence: 99%

Nitration of Tau Protein Is Linked to Neurodegeneration in Tauopathies

Horiguchi

Uryu

Giasson

et al. 2003

The American Journal of Pathology

182

118

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“…Protein concentrations were determined by staining with amido black as described [49], using bovine serum albumin as the standard. DNA in chromatin was measured by the modified diphenylamine reaction of [50] using calf thymus DNA as the standard.…”

Section: Analytical Proceduresmentioning

confidence: 99%

Conserved epitopes on plant H1 histones recognized by monoclonal antibodies

Mazzolini¹,

Vaeck²,

Montagu³

1989

European Journal of Biochemistry

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A series of monoclonal antibodies specific for distinct regions of HI histone from the plant Nicotiana tabacum were obtained from fusion experiments with spleen cells of mice immunized with tobacco nuclear extracts. These monoclonal antibodies were characterized and the evolutionary conservation of the epitopes in higher plants and animals studied by immunoblotting and enzyme-lfnked immunosorbent assay (ELISA). Whereas some epitopes appear restricted to the Solanaceae plant family, others are common to all higher eukaryotes tested and even detectable on nuclear proteins of yeast. ELISA experiments performed with isolated tobacco chromatin give some indications of the differential accessibility of the epitopes after interaction of H1 histone with the nucleosome.Inside the chromatin of eukaryotic cells the first order of DNA packing is reached by wrapping the DNA around a protein octamer composed of two molecules of histones 2A, 2B, 3, and 4 [I]. This protein-DNA complex, or nucleosome, has been found in all eukaryotic organisms studied up to now and shows in each case highly conserved structural characteristics [2]. In addition to the four nucleosomal histones, a fifth group exists which is characterized by a high lysine and arginine content. These H1 histones associate with the DNA at a point where it enters and leaves the nucleosome [3]. Furthermore, H 1 histone, once bound, aids in condensation of the nucleosome chains and in the formation and maintenance of higher-order structures in the chromatin [4, 51.Studies on the evolutionary conservation of histone proteins have shown that histones involved in nucleosome assembly are among the most conserved proteins found in eukaryotes [6 -111.In contrast to the nucleosomal histones, H1 histones show considerable sequence variation [9]. Even in a single tissue several subtypes of H1 coexist [6]. Furthermore, tissue-specific as well as development-specific HI-histone variants have been characterized [12, 131. Some variants show different abilities to condense DNA and chromatin [14 -161 which may play a role in the control of gene expression [12].Despite the significant variations in molecular mass and amino acid sequence existing among H1 histones, studies in animals revealed that the general organization of the molecule is preserved, and consists of a central hydrophobic globular domain flanked by one highly variable N-terminal domain and a hydrophilic C-terminal region [12].Only two reports on the amino acid sequence of plant H1 histones have been published until now [17, 181. The data indicate that the general structure of H1 histones has been maintained in plants. However, the amino acid sequence data remain too fragmentary to draw conclusions on the extent and modalities of evolutionary conservation of H1 histones in plants.An alternative to amino acid sequence comparisons to determine the conservation of specific structures in a protein is the use of antibodies that recognize discrete epitopes. Such an approach has been widely used in the structural characteriza...

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“…A comparison of the proteinase with the heat-induced sulfate proteinases in tomato [28], cathepsin B in CHO cells [17], as well as other heat-induced proteinases [7], [10], [11], showed that the heat-induced 65 kDa ATP-BPase of C6 cells has no similarity with them in their N-terminal 1-9 amino acid sequence, pH optimum, molecular weight and the effect of inhibitors. But it was reported that the heat shock (44 for 1 h and recovery for 1 h) could induce Neurospora crassa to express a 65 kDa ATP-BPase [20]. Its heat inducibility and other characteristics are similar to the 65 kDa ATP-BPase in C6 cells, but the exact relation of these two ATP-BPases is not known.…”

Section: Discussionmentioning

confidence: 99%

“…1 M Tris, pH 7.2, and harvested in the same buffer. Cells were disrupted by sonication (5 W, 15 sec) and the amount of protein in the homogenate determined by the method of Neuhoff et al [20]. Equal amounts of protein were then separated electrophoretically.…”

Section: Native Proteinase Gelsmentioning

confidence: 99%

Heat shock induction of a 65 kDa ATP-binding proteinase in rat C6 glioma cells

Zhang

Techel

et al. 1999

Cell Res

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The 45, 55, 65 and 100 kDa ATP-binding proteinases (ATPBPases) of the heat-shocked (44 for 30 min, recovery for 12 h) rat C6 glioma cells were purified by DEAE-ionexchange and ATP-affinity chromatography. Their molecular masses, isoelectric points (pI), pH-optima and other properties were analyzed by native proteinase gels. It was shown that the 65 kDa ATPBPase is specifically induced by heat shock and not detectable in control cells. Its N-terminal 1-9 amino acid sequence was determined by Edman degradation, but no homologies to other proteins in the protein data bases were found. 30 and 31 kDa proteinases can be cleaved from the 45, 55 and 65 kDa proteinases to which they are linked. A possible relationship of the heat-induced 65 kDa ATP-BPase with the ATP-dependent proteinases (ATP-DPases) in prokaryotes and eukaryotes is discussed. 136

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A Simple, Versatile, Sensitive and Volume-Independent Method for Quantitative Protein Determination which is Independent of Other External Influences

Cited by 234 publications

References 0 publications

Nitration of Tau Protein Is Linked to Neurodegeneration in Tauopathies

Nitration of Tau Protein Is Linked to Neurodegeneration in Tauopathies

Conserved epitopes on plant H1 histones recognized by monoclonal antibodies

Heat shock induction of a 65 kDa ATP-binding proteinase in rat C6 glioma cells

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