Subcellular fractionation, electromigration analysis and mapping of organelles

Pasquali, Christian; Fialka, Irene; Huber, Lukas A.

doi:10.1016/s0378-4347(98)00314-4

Cited by 140 publications

(121 citation statements)

References 59 publications

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“…Cells were fractionated using a ball bearing homogenizer (8.002-mm ball, HGM Laboratory Equipment) until approximately 95% of the cells were broken. Membranes were fractionated using the method described by Pasquali et al (10). The fractionated cells were centrifuged at 3,000 ϫ g for 10 min at 4°C, and the postnuclear supernatant was layered onto a 60% sucrose cushion and centrifuged at 100,000 ϫ g for 45 min.…”

Section: Preparation Of Membrane Fractions and Protein Separation-mentioning

confidence: 99%

Comprehensive Proteomic Analysis of Breast Cancer Cell Membranes Reveals Unique Proteins with Potential Roles in Clinical Cancer

Adam

Boyd

Tyson

et al. 2003

Journal of Biological Chemistry

199

167

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Proteins associated with cancer cell plasma membranes are rich in known drug and antibody targets as well as other proteins known to play key roles in the abnormal signal transduction processes required for carcinogenesis. We describe here a proteomics process that comprehensively annotates the protein content of breast tumor cell membranes and defines the clinical relevance of such proteins. Tumor-derived cell lines were used to ensure an enrichment for cancer cell-specific plasma membrane proteins because it is difficult to purify cancer cells and then obtain good membrane preparations from clinical material. Multiple cell lines with different molecular pathologies were used to represent the clinical heterogeneity of breast cancer. Peptide tandem mass spectra were searched against a comprehensive data base containing known and conceptual proteins derived from many public data bases including the draft human genome sequences. This plasma membrane-enriched proteome analysis created a data base of more than 500 breast cancer cell line proteins, 27% of which were of unknown function. The value of our approach is demonstrated by further detailed analyses of three previously uncharacterized proteins whose clinical relevance has been defined by their unique cancer expression profiles and the identification of proteinbinding partners that elucidate potential functionality in cancer.

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Section: Preparation Of Membrane Fractions and Protein Separation-mentioning

confidence: 99%

Comprehensive Proteomic Analysis of Breast Cancer Cell Membranes Reveals Unique Proteins with Potential Roles in Clinical Cancer

Adam

Boyd

Tyson

et al. 2003

Journal of Biological Chemistry

199

167

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show abstract

“…Plates were ice-chilled and the cells were washed with ice-cold PBS. Cells were scraped into ice-cold buffer A (20 mM Hepes pH 7$5, 0$15 M NaCl, 250 mM sucrose) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM pepstatin A, 1 mM leupeptine, 5 mM EDTA, 3 mM EGTA, 50 mM b-glycerophosphate, 10 mM sodium fluoride, 0$1 mM orthovanadate, and 100 nM okadaic acid) and 1 mg/ml of cycloheximide and a postnuclear supernatant was prepared as previously described (Pasquali et al 1999). The fractionation procedure was carried out on a 10-40% continuous sucrose gradient as described by Fialka et al (1997).…”

Section: Subcellular Fractionationmentioning

confidence: 99%

G protein-coupled receptor kinase 2 and β-arrestins are recruited to FSH receptor in stimulated rat primary Sertoli cells

Marion

Kara

Crépieux

et al. 2006

Journal of Endocrinology

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FSH-receptor (FSH-R) signaling is regulated by agonistinduced desensitization and internalization. It has been shown, in a variety of overexpression systems, that G protein-coupled receptor kinases (GRKs) phosphorylate the activated FSH-R, promote b-arrestin recruitment and ultimately lead to internalization. The accuracy of this mechanism has not yet been demonstrated in cells expressing these different molecules at physiological levels. Using sucrose gradient fractionation, we show that FSH induces the recruitment of the endogenous GRK 2 and b-arrestin 1/2 from the cytoplasm to the plasma membrane of rat primary Sertoli cells. As assessed by ligand binding, the FSH-R was found expressed in the fractions where GRK 2 and b-arrestins were recruited upon FSH treatment. In addition, the endogenous b-arrestin 1 was found dephosphorylated in an agonist-dependent manner. Finally, a significant FSH-binding activity was co-immunoprecipitated with the endogenous b-arrestins from agonist-stimulated but not from untreated Sertoli cell extracts. This FSH-R interaction with b-arrestins was sustained for up to 30 min. In conclusion, our data strongly suggest that the GRK/b-arrestin machinery plays a physiologically relevant role in the regulation of the FSH signaling.

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“…bacteria and subcellular organelles, is becoming increasingly important in biomedicine and in environmental and food analysis. 1,2 Methods such as blood culture of bacteria 1 and subcellular fractionation 3 are, however, laborintensive, complicated, and time-consuming, and new technologies are being sought to redress these shortcomings. Microfluidics offers a means of automated handling and analysis of sub-micrometer bioparticles with the associated advantage of a continuous mode of sample handling.…”

Section: Introductionmentioning

confidence: 99%

Focusing of sub-micrometer particles and bacteria enabled by two-dimensional acoustophoresis

et al. 2014

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Subcellular fractionation, electromigration analysis and mapping of organelles

Cited by 140 publications

References 59 publications

Comprehensive Proteomic Analysis of Breast Cancer Cell Membranes Reveals Unique Proteins with Potential Roles in Clinical Cancer

Comprehensive Proteomic Analysis of Breast Cancer Cell Membranes Reveals Unique Proteins with Potential Roles in Clinical Cancer

G protein-coupled receptor kinase 2 and β-arrestins are recruited to FSH receptor in stimulated rat primary Sertoli cells

Focusing of sub-micrometer particles and bacteria enabled by two-dimensional acoustophoresis

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