Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity

Morrissey, James H.

doi:10.1016/0003-2697(81)90783-1

Cited by 4,018 publications

(1,455 citation statements)

References 7 publications

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“…The fragments were analysed on 11% SDS-PAGE gels and then detected by silver staining. 36 Mass spectrometry. The caspase cleavage assay was scaled up and analysed on a Coomassie stained SDS-PAGE gel.…”

Section: Methodsmentioning

confidence: 99%

Cleavage and degradation of Claspin during apoptosis by caspases and the proteasome

et al. 2007

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Apoptosis plays a crucial role in development and tissue homeostasis. Some key survival pathways, such as DNA damage checkpoints and DNA repair, have been described to be inactivated during apoptosis. Here, we describe the processing of the human checkpoint protein Claspin during apoptosis. We observed cleavage of Claspin into multiple fragments in vivo. In vitro cleavage with caspases 3 and 7 of various fragments of the protein, revealed cut sites near the N-and C-termini of the protein.Using mass spectrometry, we identified a novel caspase cleavage site in Claspin at Asp25. Importantly, in addition to cleavage by caspases, we observed a proteasome-dependent degradation of Claspin under apoptotic conditions, resulting in a reduction of the levels of both full-length Claspin and its cleavage products. This degradation was not dependent upon the DSGxxS phosphodegron motif required for SCF b-TrCP -mediated ubiquitination of Claspin. Finally, downregulation of Claspin protein levels by short interfering RNA resulted in an increase in apoptotic induction both in the presence and absence of DNA damage. We conclude that Claspin has antiapoptotic activity and is degraded by two different pathways during apoptosis. The resulting disappearance of Claspin from the cells further promotes apoptosis.

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Section: Methodsmentioning

confidence: 99%

Cleavage and degradation of Claspin during apoptosis by caspases and the proteasome

et al. 2007

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“…After IEF, IEF gels were set on top of a linear gradient 10-13% SDS polyacrylamide gel and electrophoresed overnight using 10 mA per gel at 10°C. After protein fixation, proteins were visualized by silver staining (Morrissey, 1981).…”

Section: Methodsmentioning

confidence: 99%

Relationship between TA01 and TA02 polypeptides associated with lung adenocarcinoma and histocytological features

Hirano¹,

Fujioka²,

Franzén³

et al. 1997

Br J Cancer

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Summary TA01 (molecular weight 35.0 kDa, isoelectric point 5.45) and TA02 (molecular weight 35.0 kDa, isoelectric point 5.29) polypeptides were detected using two-dimensional polyacrylamide gel electrophoresis (2-DE). A previous study has shown that these polypeptides are distributed in primary adenocarcinomas and some large -cell carcinomas of the lung. However, various expression levels of TA01 and TA02 polypeptides were demonstrated in adenocarcinoma, while large-cell carcinoma expressed low levels. To evaluate the relationship between the expression of TA01 and TA02 polypeptides and the histocytological features of primary adenocarcinoma of the lung, these two polypeptides were analysed by 2-DE combined with a non-enzymatic sample preparation technique, and their expression levels were compared with the histocytological features of primary lung adenocarcinoma. Out of 57 primary lung adenocarcinoma cases, 46 cases (80.7%) and 52 cases (91.2%) expressed TA01 and TA02 polypeptides respectively. Furthermore, the expression levels of TA01 and TA02 polypeptides correlated with the degree of cellular atypia, structural atypia and histocytological differentiation of primary lung adenocarcinoma. On the other hand, these two polypeptides were not detected in adenocarcinoma of the lung, metastatic from the colon and mammary glands. High expression of TA01 and TA02 polypeptides reflected the differentiation of primary adenocarcinoma in the lung. These two polypeptides are valuable in determining the histocytological differentiation of primary lung adenocarcinoma as well as in distinguishing between primary and metastatic adenocarcinoma of the lung.Keywords: primary adenocarcinoma of the lung; histopathological differentiation; TA01 polypeptide; TA02 polypeptide; two-dimensional polyacrylamide gel electrophoresis Tumour markers can contribute to clinicopathological diagnosis, more accurate evaluation of biological malignancy of the tumour and selection of therapeutic strategy. Products of oncogenes and mutant tumour-suppressor genes are possible candidates for biological tumour markers. Also, histological differentiation antigens could be useful markers in cases of borderline histology.Although some tumour markers (e.g. CEA, CA 19-9 and TPA, etc.) are used in the clinical diagnosis of primary adenocarcinoma of the lung (Vincent et al, 1979;Latanche et al, 1985;Buccheri et al, 1988;Niklinski et al, 1995), there is no specific tumour marker for primary adenocarcinoma of the lung. At present, cell proliferation and products of oncogenes and tumour-suppressor genes in lung adenocarcinoma are attracting attention (M0rkve et al, 1992;Scagliotti et al, 1993;Ebina et al, 1994;Rachwal et al, 1995;Costa et al, 1996) Correspondence to: T Hirano Linnoila et al, 1992) and protein 1 (Barnard et al, 1992). There is a possibility that these polypeptides become specific tumour markers for primary adenocarcinoma of the lung. Such a marker might be useful to distinguish primary and metastatic adenocarcinoma of the lung.We also investigate...

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“…SDS-PAGE gels (15%) were run on a Hoefer Mighty Small II apparatus or a BRL model V16 electrophoresis system. Silver staining was performed according to the protocol of Morrissey (1981) with minor modifications (Jones et al, 1987). Proteins on the gel were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) or nitrocellulose membranes using a Hoefer Transblot apparatus.…”

Section: Electrophoresis Immunoblotting and Autoradiographymentioning

confidence: 99%

The FliP and FliR proteins of Salmonella typhimurium, putative components of the type III flagellar export apparatus, are located in the flagellar basal body

Fan

Ohnishi

Francis

et al. 1997

Molecular Microbiology

120

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SummaryMost of the structural components of the flagellum of Salmonella typhimurium are exported through a flagellum-specific pathway, which is a member of the family of type III secretory pathways. The export apparatus for this process is poorly understood. A previous study has shown that two proteins, about 23 and 26 kDa in size and of unknown genetic origin, are incorporated into the flagellar basal body at a very early stage of flagellar assembly. In the present study, we demonstrate that these basal body proteins are FliP (in its mature form after signal peptide cleavage) and FliR respectively. Both of these proteins have homologues in other type III secretion systems. By placing a FLAG epitope tag on FliR and the MS-ring protein FliF and immunoblotting isolated hook basal body complexes with anti-FLAG monoclonal antibody, we estimate (using the FLAG-tagged FliF as an internal reference) that the stoichiometry of FliR is fewer than three copies per basal body. An independent estimate of stoichiometry was made using data from an earlier quantitative radiolabelling analysis, yielding values of around four or five subunits per basal body for FliP and around one subunit per basal body for FliR. Immunoelectron microscopy using anti-FLAG antibody and gold-protein A suggests that FliR is located near the MS ring. We propose that the flagellar export apparatus contains FliP and FliR and that this apparatus is embedded in a patch of membrane in the central pore of the MS ring.

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Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity

Cited by 4,018 publications

References 7 publications

Cleavage and degradation of Claspin during apoptosis by caspases and the proteasome

Cleavage and degradation of Claspin during apoptosis by caspases and the proteasome

Relationship between TA01 and TA02 polypeptides associated with lung adenocarcinoma and histocytological features

The FliP and FliR proteins of Salmonella typhimurium, putative components of the type III flagellar export apparatus, are located in the flagellar basal body

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