A cost-effective device for the rapid transfer of gel-separated proteins onto membranes

Tam, Hann W.; Huang, Yu‐Chen; Tam, Ming F.

doi:10.1016/j.ab.2008.11.028

Cited by 2 publications

(2 citation statements)

References 5 publications

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“…Proteins in the samples were quantified using a DC Protein Assay Kit (Bio‐Rad, Hercules, CA, USA) and separated electrophoretically on precast 4–20% NuPAGE gels (Invitrogen, Carlsbad, CA, USA) under denaturing conditions. Proteins were visualized by the Blue Silver dye staining method (Candiano et al , 2004) or transferred onto polyvinylidene difluoride membranes (Tam et al , 2009) for western analysis according to Sadra et al (2009), using antibodies against Rpl12p and Stm1p (control), respectively. Rabbit antibodies against Stm1p were raised in our laboratory using bacterial‐expressed recombinant proteins as antigens.…”

Section: Methodsmentioning

confidence: 99%

EPR Characterisation of the Ferrous Nitrosyl Complex Formed within the Oxygenase Domain of NO Synthase

et al. 2013

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Nitric oxide is produced in mammals by a class of enzymes called NO synthases (NOSs). It plays a central role in cellular signalling but also has deleterious effects, as it leads to the production of reactive oxygen and nitrogen species. NO forms a relatively stable adduct with ferrous haem proteins, which, in the case of NOS, is also a key catalytic intermediate. Despite extensive studies on the ferrous nitrosyl complex of other haem proteins (in particular myoglobin), little characterisation has been performed in the case of NOS. We report here a temperature-dependent EPR study of the ferrous nitrosyl complex of the inducible mammalian NOS and the bacterial NOS-like protein from Bacillus subtilis. The results show that the overall behaviours are similar to those observed for other haem proteins, but with distinct ratios between axial and rhombic forms in the case of the two NOS proteins. The distal environment appears to control the existence of the axial form and the evolution of the rhombic form.

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Section: Methodsmentioning

confidence: 99%

EPR Characterisation of the Ferrous Nitrosyl Complex Formed within the Oxygenase Domain of NO Synthase

et al. 2013

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“…Proteins in the samples were quantified using a DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA) and separated electrophoretically on precast 4-20% NuPAGE gels (Invitrogen, Carlsbad, CA, USA) under denaturing conditions. Proteins were visualized by the Blue Silver dye staining method (Candiano et al, 2004) or transferred onto polyvinylidene difluoride membranes (Tam et al, 2009) for western analysis according to Sadra et al (2009), using antibodies against Rpl12p and Stm1p (control), respectively. Rabbit antibodies against Stm1p were raised in our laboratory using bacterial-expressed recombinant proteins as antigens.…”

Section: Western Blot Analysismentioning

confidence: 99%

Rpl12p affects the transcription of the PHO pathway high‐affinity inorganic phosphate transporters and repressible phosphatases

Huang

Liu

et al. 2011

Yeast

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The ribosomal protein Rpl12p of Saccharomyces cerevisiae is encoded by duplicated genes, RPL12A and RPL12B. The gene products possess an identical amino acid sequence. Yeast strain 6EA1, which lacks both genes, is viable but exhibits a very slow-growth phenotype. In this study, 6EA1 cells were transformed with plasmids carrying either RPL12A or RPL12B, and the transcriptional profiles of wild-type W303, 6EA1 and the transformed cells grown in synthetic complete medium were examined by microarray analysis. Transcription of PHO84, a gene encoding a high-affinity phosphate transporter, was drastically suppressed in 6EA1. PHO84 expression is induced under phosphate-limiting conditions. Therefore, cells were grown in low-phosphate medium and transcripts encoding the PHO pathway proteins were quantified by qRT-PCR. The high-affinity phosphate transporters and repressible phosphatases were suppressed, while PHO4, a PHO pathway transcription activator, was upregulated in 6EA1. Accordingly, phosphate transport and acidic phosphatase activities were significantly decreased in 6EA1. Addition of RPL12A or RPL12B to 6EA1 largely lessens these effects. We postulate that RPL12 has an extra-ribosomal function in modulating the transcription of genes that need Pho4p activation.

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A cost-effective device for the rapid transfer of gel-separated proteins onto membranes

Cited by 2 publications

References 5 publications

EPR Characterisation of the Ferrous Nitrosyl Complex Formed within the Oxygenase Domain of NO Synthase

EPR Characterisation of the Ferrous Nitrosyl Complex Formed within the Oxygenase Domain of NO Synthase

Rpl12p affects the transcription of the PHO pathway high‐affinity inorganic phosphate transporters and repressible phosphatases

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