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Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

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Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins. / Königshausen, Eva; Potthoff, Sebastian A; Haase, Raphael; Meyer-Schwesinger, Catherine; Kaufmann, Ernest; Rump, L Christian; Stegbauer, Johannes; Sellin, Lorenz; Quack, Ivo; Woznowski, Magdalena.

In: JOVE-J VIS EXP, No. 143, 18.01.2019.

Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review

Harvard

Königshausen, E, Potthoff, SA, Haase, R, Meyer-Schwesinger, C, Kaufmann, E, Rump, LC, Stegbauer, J, Sellin, L, Quack, I & Woznowski, M 2019, 'Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins', JOVE-J VIS EXP, no. 143. https://doi.org/10.3791/58542

APA

Königshausen, E., Potthoff, S. A., Haase, R., Meyer-Schwesinger, C., Kaufmann, E., Rump, L. C., Stegbauer, J., Sellin, L., Quack, I., & Woznowski, M. (2019). Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins. JOVE-J VIS EXP, (143). https://doi.org/10.3791/58542

Vancouver

Königshausen E, Potthoff SA, Haase R, Meyer-Schwesinger C, Kaufmann E, Rump LC et al. Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins. JOVE-J VIS EXP. 2019 Jan 18;(143). https://doi.org/10.3791/58542

Bibtex

@article{42337cd5851947299ee2f2355090a46a,
title = "Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins",
abstract = "Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).",
keywords = "Journal Article, Video-Audio Media",
author = "Eva K{\"o}nigshausen and Potthoff, {Sebastian A} and Raphael Haase and Catherine Meyer-Schwesinger and Ernest Kaufmann and Rump, {L Christian} and Johannes Stegbauer and Lorenz Sellin and Ivo Quack and Magdalena Woznowski",
year = "2019",
month = jan,
day = "18",
doi = "10.3791/58542",
language = "English",
journal = "JOVE-J VIS EXP",
issn = "1940-087X",
publisher = "MYJoVE Corporation",
number = "143",

}

RIS

TY - JOUR

T1 - Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

AU - Königshausen, Eva

AU - Potthoff, Sebastian A

AU - Haase, Raphael

AU - Meyer-Schwesinger, Catherine

AU - Kaufmann, Ernest

AU - Rump, L Christian

AU - Stegbauer, Johannes

AU - Sellin, Lorenz

AU - Quack, Ivo

AU - Woznowski, Magdalena

PY - 2019/1/18

Y1 - 2019/1/18

N2 - Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

AB - Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

KW - Journal Article

KW - Video-Audio Media

U2 - 10.3791/58542

DO - 10.3791/58542

M3 - SCORING: Journal article

C2 - 30735184

JO - JOVE-J VIS EXP

JF - JOVE-J VIS EXP

SN - 1940-087X

IS - 143

ER -