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Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine

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Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine. / Krebs, Christian; Koestner, Wolfgang; Nissen, Marion; Welge, Vivienne; Parusel, Ines; Malavasi, Fabio; Leiter, Edward H; Santella, Regina M; Haag, Friedrich; Koch-Nolte, Friedrich.

in: ANAL BIOCHEM, Jahrgang 314, Nr. 1, 01.03.2003, S. 108-15.

Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung

Harvard

Krebs, C, Koestner, W, Nissen, M, Welge, V, Parusel, I, Malavasi, F, Leiter, EH, Santella, RM, Haag, F & Koch-Nolte, F 2003, 'Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine', ANAL BIOCHEM, Jg. 314, Nr. 1, S. 108-15.

APA

Krebs, C., Koestner, W., Nissen, M., Welge, V., Parusel, I., Malavasi, F., Leiter, E. H., Santella, R. M., Haag, F., & Koch-Nolte, F. (2003). Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine. ANAL BIOCHEM, 314(1), 108-15.

Vancouver

Krebs C, Koestner W, Nissen M, Welge V, Parusel I, Malavasi F et al. Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine. ANAL BIOCHEM. 2003 Mär 1;314(1):108-15.

Bibtex

@article{0bd16962c4344c408648c7047e7a2254,
title = "Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine",
abstract = "NAD-dependent ADP-ribosylation is one of the posttranslational protein modifications. On mammalian cells, glycosylphosphatidylinositol-anchored cell surface ADP-ribosyltransferases (ARTs) ADP-ribosylate other cell surface proteins and thereby affect important cellular functions. Here we describe convenient flow-cytometric and immunoblot assays for monitoring ADP-ribosylation of cell surface proteins on living cells by exploiting the capacity of ARTs to utilize etheno-NAD as substrate. Etheno-ADP-ribosylation of cell surface proteins can be detected by flow cytometry with 1G4, a monoclonal antibody specific for ethenoadenosine. Labeling of cells with 1G4 is dependent on the expression of cell surface ARTs and occurs only after incubation of ART-expressing cells with etheno-NAD and not with etheno-ADP-ribose. Dose-response analyses show efficient 1G4 staining of ART-expressing cells at micromolar etheno-NAD concentrations. Half-maximal staining is obtained with 1-2 micro M etheno-NAD, saturation is reached at 5-20 micro M etheno-NAD. Immunoblot analyses confirm that ART-expressing cells incorporate ethenoadenosine covalently (i.e., SDS resistant) into several cell surface proteins. The flow-cytometric 1G4 staining assay can be used to identify subpopulations of cells expressing cell surface ART activity and to select ART(hi) cell variants. The immunoblot 1G4 staining assay can also be used to identify etheno-ADP-ribosylated target proteins. These new assays hold promise for many interesting applications in biochemistry and cell biology.",
keywords = "ADP Ribose Transferases, Adenosine, Animals, Antibodies, Monoclonal, Ethylenes, Flow Cytometry, Humans, Immunoblotting, Membrane Proteins, Mice, Mice, Inbred C57BL, Tumor Cells, Cultured, Journal Article",
author = "Christian Krebs and Wolfgang Koestner and Marion Nissen and Vivienne Welge and Ines Parusel and Fabio Malavasi and Leiter, {Edward H} and Santella, {Regina M} and Friedrich Haag and Friedrich Koch-Nolte",
year = "2003",
month = mar,
day = "1",
language = "English",
volume = "314",
pages = "108--15",
journal = "ANAL BIOCHEM",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "1",

}

RIS

TY - JOUR

T1 - Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine

AU - Krebs, Christian

AU - Koestner, Wolfgang

AU - Nissen, Marion

AU - Welge, Vivienne

AU - Parusel, Ines

AU - Malavasi, Fabio

AU - Leiter, Edward H

AU - Santella, Regina M

AU - Haag, Friedrich

AU - Koch-Nolte, Friedrich

PY - 2003/3/1

Y1 - 2003/3/1

N2 - NAD-dependent ADP-ribosylation is one of the posttranslational protein modifications. On mammalian cells, glycosylphosphatidylinositol-anchored cell surface ADP-ribosyltransferases (ARTs) ADP-ribosylate other cell surface proteins and thereby affect important cellular functions. Here we describe convenient flow-cytometric and immunoblot assays for monitoring ADP-ribosylation of cell surface proteins on living cells by exploiting the capacity of ARTs to utilize etheno-NAD as substrate. Etheno-ADP-ribosylation of cell surface proteins can be detected by flow cytometry with 1G4, a monoclonal antibody specific for ethenoadenosine. Labeling of cells with 1G4 is dependent on the expression of cell surface ARTs and occurs only after incubation of ART-expressing cells with etheno-NAD and not with etheno-ADP-ribose. Dose-response analyses show efficient 1G4 staining of ART-expressing cells at micromolar etheno-NAD concentrations. Half-maximal staining is obtained with 1-2 micro M etheno-NAD, saturation is reached at 5-20 micro M etheno-NAD. Immunoblot analyses confirm that ART-expressing cells incorporate ethenoadenosine covalently (i.e., SDS resistant) into several cell surface proteins. The flow-cytometric 1G4 staining assay can be used to identify subpopulations of cells expressing cell surface ART activity and to select ART(hi) cell variants. The immunoblot 1G4 staining assay can also be used to identify etheno-ADP-ribosylated target proteins. These new assays hold promise for many interesting applications in biochemistry and cell biology.

AB - NAD-dependent ADP-ribosylation is one of the posttranslational protein modifications. On mammalian cells, glycosylphosphatidylinositol-anchored cell surface ADP-ribosyltransferases (ARTs) ADP-ribosylate other cell surface proteins and thereby affect important cellular functions. Here we describe convenient flow-cytometric and immunoblot assays for monitoring ADP-ribosylation of cell surface proteins on living cells by exploiting the capacity of ARTs to utilize etheno-NAD as substrate. Etheno-ADP-ribosylation of cell surface proteins can be detected by flow cytometry with 1G4, a monoclonal antibody specific for ethenoadenosine. Labeling of cells with 1G4 is dependent on the expression of cell surface ARTs and occurs only after incubation of ART-expressing cells with etheno-NAD and not with etheno-ADP-ribose. Dose-response analyses show efficient 1G4 staining of ART-expressing cells at micromolar etheno-NAD concentrations. Half-maximal staining is obtained with 1-2 micro M etheno-NAD, saturation is reached at 5-20 micro M etheno-NAD. Immunoblot analyses confirm that ART-expressing cells incorporate ethenoadenosine covalently (i.e., SDS resistant) into several cell surface proteins. The flow-cytometric 1G4 staining assay can be used to identify subpopulations of cells expressing cell surface ART activity and to select ART(hi) cell variants. The immunoblot 1G4 staining assay can also be used to identify etheno-ADP-ribosylated target proteins. These new assays hold promise for many interesting applications in biochemistry and cell biology.

KW - ADP Ribose Transferases

KW - Adenosine

KW - Animals

KW - Antibodies, Monoclonal

KW - Ethylenes

KW - Flow Cytometry

KW - Humans

KW - Immunoblotting

KW - Membrane Proteins

KW - Mice

KW - Mice, Inbred C57BL

KW - Tumor Cells, Cultured

KW - Journal Article

M3 - SCORING: Journal article

C2 - 12633608

VL - 314

SP - 108

EP - 115

JO - ANAL BIOCHEM

JF - ANAL BIOCHEM

SN - 0003-2697

IS - 1

ER -