Production of Antipolyubiquitin Monoclonal Antibodies and Their Use for Characterization and Isolation of Polyubiquitinated Proteins

doi:10.1016/S0076-6879(05)99006-X

Methods in Enzymology

Volume 399, 2005, Pages 75-86

https://doi.org/10.1016/S0076-6879(05)99006-X Get rights and content

Abstract

Formation of a Lys48‐linked polyubiquitin chain is required for destruction of targeted proteins by the 26S proteasome, whereas formation of a Lys63‐linked polyubiquitin chain is required for modulation of protein–protein interaction, enzyme activity, and intracellular localization. In addition, monoubiquitination plays key roles in endocytosis and protein trafficking. To gain a better understanding of the role of polyubiquitination, we attempted to produce monoclonal antibodies against the polyubiquitin chains, two of which were designated as FK1 and FK2 and were extensively characterized. Both FK1 and FK2 antibodies recognize the polyubiquitin moiety but not free ubiquitin, whereas FK2 antibody, but not FK1 antibody, can recognize monoubiquitinated proteins. The FK1/FK2 antibodies can be applied to ELISA for quantification of polyubiquitin chains, to immunocytochemistry for staining of intracellular polyubiquitin chains, and also to immunoaffinity chromatography for isolation of polyubiquitinated proteins. Thus, these two antibodies are useful for isolating polyubiquitin chain–tagged proteins and for probing proteins that are modified through polyubiquitination or monoubiquitination in various cells and tissues under physiological and pathological conditions.

Introduction

Posttranslational protein modifications play key roles in the regulation of cellular events. Ubiquitin‐conjugation (i.e., modification with polyubiquitin chains or monoubiquitin) is involved in various cellular events, including cell cycle progression, signal transduction, endocytosis, transcription, DNA repair, apoptosis, and immune response (Glickman 2001, Hershko 1998, Hicke 2001, Pickart 2001). Polyubiquitin chains linked through Lys48 function as a signal for degradation of target proteins by the 26S proteasome; chains linked through Lys63 are involved in proteasome‐independent events such as DNA repair, endocytosis, and signal transduction; and monoubiquitination is involved in endocytosis and protein trafficking. We succeeded in producing several monoclonal antibodies against the polyubiquitin chains, including FK1 and FK2 antibodies (Fujimuro et al., 1994). The FK1 and FK2 antibodies recognize the polyubiquitin moiety but not free ubiquitin, and FK2 antibody shows affinity to proteins tagged with monoubiquitin. Both antibodies have been applied to ELISA for quantification of polyubiquitin chains (Takada et al., 1995), to immunocytochemistry for staining of intracellular polyubiquitin chains (Fujimuro et al., 1997), and to immunoaffinity chromatography for isolation of ubiquitin–protein conjugates and thioester‐linked ubiquitin‐intermediates (Takada et al., 2001). Thus, these antibodies are useful tools for the identification, purification, and quantification of polyubiquitinated proteins and polyubiquitin chains that have been formed in various cells and tissues under physiological and pathological conditions. In this chapter, we describe in detail methods for the production of antipolyubiquitin monoclonal antibodies and their use for characterization and isolation of polyubiquitinated proteins.

Section snippets

Production of Antipolyubiquitin Monoclonal Antibodies

We prepared ubiquitin‐conjugated proteins using a partial purified enzyme mixture of E1, E2s, and E3s, and we produced antipolyubiquitin monoclonal antibodies using the thus‐prepared polyubiquitin‐conjugated proteins as antigen (Fujimuro et al., 1994).

Characterization of Polyubiquitin Chains by the Use of Antipolyubiquitin Monoclonal Antibodies

The use of immunoblotting and immunofluorescent staining with FK1 and FK2 antibodies has been reported to enable detection of dynamic change in the amount, localization, and distribution of the ubiquitin‐conjugate moiety (for example, see Lelouard 2002, Sawada 2002, Schubert 2000), as well as identification of ubiquitin‐conjugates (for example, see Takada et al. [2001]). Furthermore, these antibodies have been used to investigate the formation of polyubiquitin conjugates under pathological

Isolation of Polyubiquitinated Proteins by the Use of Antipolyubiquitin Monoclonal Antibodies

FK2 antibody (IgG1) is more suitable than FK1 antibody for use in immunoprecipitation together with protein A/G‐Sepharose beads, because the subclass of FK1 antibody is IgM. Western blotting and ELISA revealed that FK2 antibody recognizes polyubiquitin and monoubiquitin conjugates but not ubiquitin molecules that had been immobilized on a nitrocellulose membrane in the immunoprecipitation or a microtiter well in the ELISA (Fujimuro 1994, Takada 1995), but it should be noted that in the

Conclusions

Antipolyubiquitin monoclonal antibodies described in this chapter are useful for isolating polyubiquitinated proteins and for probing proteins that are modified through polyubiquitination or monoubiquitination under various conditions. As described in yeast nin1‐1 cells, impairment of the 26S proteasome results in the accumulation of polyubiquitinated proteins, and, conversely, when the accumulation of polyubiquitinated proteins, which can be detected only by antipolyubiquitin monoclonal

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Antibodies as means for selective mass spectrometry
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Production of Antipolyubiquitin Monoclonal Antibodies and Their Use for Characterization and Isolation of Polyubiquitinated Proteins

Abstract

Introduction

Section snippets

Production of Antipolyubiquitin Monoclonal Antibodies

Characterization of Polyubiquitin Chains by the Use of Antipolyubiquitin Monoclonal Antibodies

Isolation of Polyubiquitinated Proteins by the Use of Antipolyubiquitin Monoclonal Antibodies

Conclusions

FEBS Lett.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

UCH‐L1 aggresome formation in response to proteasome impairment indicates a role in inclusion formation in Parkinson's disease

J. Neurochem.

Nuclear sequestration of cellular chaperone and proteasomal machinery during herpes simplex virus type 1 infection

J. Virol.

Dynamics of ubiquitin conjugation during heat‐shock response revealed by using a monoclonal antibody specific to multi‐ubiquitin chains

Eur. J. Biochem.

The ubiquitin‐proteasome proteolytic pathway: Destruction for the sake of construction

Physiol. Rev.

Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation

Nature Cell Biol.

The ubiquitin system

Annu. Rev. Biochem.