SDS disc electrophoresis of proteins in homogeneous, low‐concentrated polyacrylamide gels

Maly, I. Piotr; Nitsch, Cordula

doi:10.1002/elps.200600688

Cited by 6 publications

(4 citation statements)

References 13 publications

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“…For controls, the primary antibody was omitted. In addition, the specifity of antibodies was tested on homogenates of mouse brain or rat liver using our high-resolution SDS electrophoresis system (Maly and Nitsch, 2007 ; Maly and Landmann, 2008 ). For detection of endogenous activity of alkaline phosphatase, cryosections were fixed in 4% paraformaldehyde in PBS at pH 7.4 for 30 min at room temperature, rinsed with AMPD buffer and incubated with BCIP/NBT alkaline phosphatase substrate system for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Claudin-1, -2 and -3 Are Selectively Expressed in the Epithelia of the Choroid Plexus of the Mouse from Early Development and into Adulthood While Claudin-5 is Restricted to Endothelial Cells

et al. 2016

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A primary function of epithelial and endothelial monolayers is the formation of barriers that separate tissues into functional compartments. Tight junctions (TJs) seal the intercellular space between the single cells of a monolayer. TJs thus contribute importantly to the homeostasis of the cerebrospinal fluid as they help in maintaining the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (CSF). The composition of TJs differs by its localization as well as the stage of development according to its respective function. Claudin-3 is typically present in the epithelia and has been claimed to be a constituent of the BBB. It is, however, notoriously difficult to demonstrate its expression in endothelial cells of the brain vasculature at the morphological level by means of immunohistochemical techniques. Using an improved fixation strategy (4% paraformaldehyde at pH 11, in the presence of EDTA) and the sensitive alkaline phosphatase as a detection system, we show that claudin-3 is present in mouse epithelia from embryonic day 14 onwards. In brain, it is restricted to the anlage of choroid plexus in the ventricles, together with claudin-1 and -2. In adult mice, it is clearly delineating the epithelium of the choroid plexus in the lateral and fourth ventricles. In contrast, in cerebral blood vessels claudin-3 as well as claudin-1 and -2 are absent in cerebral blood vessels during all developmental stages up to adulthood. Rather, the BBB is characterized by the presence of claudin-5, ZO-1 and occludin. Thus, in mice claudin-3 is an important constituent of TJ in the embryonic and in the adult choroid plexus.

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Section: Methodsmentioning

confidence: 99%

Claudin-1, -2 and -3 Are Selectively Expressed in the Epithelia of the Choroid Plexus of the Mouse from Early Development and into Adulthood While Claudin-5 is Restricted to Endothelial Cells

et al. 2016

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“…Electrophoresis on 0.35-mm thick SDS-polyacrylamide slab gels (4%) bound to glass plates was performed as described (Maly and Nitsch 2007). Samples were transferred for 16 h from ultra thin-layer SDS polyacrylamide gels to nitrocellulose membranes (0.2 m pore size, Bio-Rad, Reinach, Switzerland) using the diVusion blotting technique.…”

Section: Polyacrylamide Gel Electrophoresis and Immunoblottingmentioning

confidence: 99%

Bile duct ligation in the rat causes upregulation of ZO-2 and decreased colocalization of claudins with ZO-1 and occludin

Maly

Landmann

2008

Histochem Cell Biol

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As the only barrier between blood and bile compartments hepatocellular tight junctions play a crucial role in cholestasis-induced increase of biliary permeability. The molecular basis of this reversible defect is not known. We, therefore, examined expression, phosphorylation, distribution and colocalization of the junctional proteins occludin, claudin-1-3, ZO-1 and ZO-2 in rats after bile duct ligation and release of ligation. In control rats, claudin-1 and ZO-2 displayed a lobular gradient with highest expression levels in periportal cells, whereas claudin-2 showed a reciprocal distribution. Other proteins were evenly expressed in the liver lobule. Ligation resulted in upregulation of ZO-2 (2.7-fold), ZO-1 (1.4-fold) and occludin (1.2-fold) but not of claudins. Only ZO-2 showed increased phosphorylation. Distribution patterns were unchanged except for a strong accumulation of ZO-2 in perivenous hepatocytes. Colocalization analysis demonstrated that perivenous ZO-2 was the only protein examined revealing strongly increased overlap with occludin and ZO-1, whereas claudins and other proteins displayed a decrease. All changes were partially reversed by release of ligation. We conclude that differential expression of claudin-1-2 and ZO-2 has functional implications for bile formation. The moderately increased ZO-1 and occludin levels account for the known elongation of tight junction strands. The highly increased expression and changed distribution of ZO-2 suggests that ZO-1 is partly substituted by ZO-2, an alteration possibly causing impaired barrier function.

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“…The stacking of the proteins occurs between the leading (chloride) ions and the trailing (glycine or tricine) ions at pH 7, a pH value, where glycine or tricine have still a lower mobility than the SDS-protein micelles. Maly and Nitsch, (2007) have taken a similar approach with taurine as trailing ion in the gel, in order to reduce the continuous stacking of SDS in the gel and get a better resolution of low molecular weight proteins. But this technique requires some handling efforts: parts of the gel have to be equilibrated in different buffers short before use.…”

Section: The Second Dimension: Sds-pagementioning

confidence: 99%

The current state of the art in high-resolution two-dimensional electrophoresis

Westermeier¹,

Schickle²

2009

Archives of Physiology and Biochemistry

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The study of the "proteomes" of human cells, tissues, and body fluids is a big challenge, and several highly sophisticated workflow approaches are pursued to achieve as comprehensive information as possible. Initially proteome analysis was exclusively based on the gel-based workflow, employing two-dimensional electrophoresis of protein extracts followed by mass spectrometry of the tryptic peptide digests of protein spots. Meanwhile several additional proteomics workflows are applied, which are mostly based on separation and analysis of tryptic peptides without separating the protein mixture. However, direct information on quantitative and qualitative changes of protein expressions can only be obtained by methods operating on the protein level, no other method can replace two-dimensional electrophoresis. In this review we compile the different techniques of high-resolution two-dimensional electrophoresis and their further developments to increase the degree of reliance of the method.

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SDS disc electrophoresis of proteins in homogeneous, low‐concentrated polyacrylamide gels

Cited by 6 publications

References 13 publications

Claudin-1, -2 and -3 Are Selectively Expressed in the Epithelia of the Choroid Plexus of the Mouse from Early Development and into Adulthood While Claudin-5 is Restricted to Endothelial Cells

Claudin-1, -2 and -3 Are Selectively Expressed in the Epithelia of the Choroid Plexus of the Mouse from Early Development and into Adulthood While Claudin-5 is Restricted to Endothelial Cells

Bile duct ligation in the rat causes upregulation of ZO-2 and decreased colocalization of claudins with ZO-1 and occludin

The current state of the art in high-resolution two-dimensional electrophoresis

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