Different biochemical techniques are well established to investigate target’s ubiquitination in mammals without overexpressing a tagged version of ubiquitin (Ub). The simplest and more direct approach is to immunoprecipitate (IP) your target protein from cell lysate (stimulated and/or properly treated), followed by western blot analysis utilizing specifi c antibodies against Ub (see Subheading 3.1). This approach requires a good antibody against the target working in IP; alternatively, one could express a tagged version of the protein, possibly at the endogenous level. Another approach consists in IP ubiquitinated proteins from total cell lysate followed by detection with the antibody against the protein of interest. This second method relies on the availability of specifi c and very effi cient antibodies against Ub (see Subheading 3.2). A more quantitative approach is the DELFIA assay (Perkin Elmer), an ELISA-based assay, which allows comparing more samples and conditions (see Subheading 3.3). Cross-validation with more than one approach is usually recommended in order to prove that your protein is modifi ed by ubiquitin. Here we will use the EGFR as model system but protocols can be easily modifi ed according to the protein of interest.

Strategies to detect endogenous ubiquitination of a target mammalian protein / S. Sigismund, S. Polo (METHODS IN MOLECULAR BIOLOGY). - In: Proteostasis / [a cura di] R. Matthiesen. - Prima edizione. - [s.l] : Humana Press Inc., 2016. - ISBN 9781493937547. - pp. 143-151

Strategies to detect endogenous ubiquitination of a target mammalian protein

S. Sigismund;S. Polo
Ultimo
2016

Abstract

Different biochemical techniques are well established to investigate target’s ubiquitination in mammals without overexpressing a tagged version of ubiquitin (Ub). The simplest and more direct approach is to immunoprecipitate (IP) your target protein from cell lysate (stimulated and/or properly treated), followed by western blot analysis utilizing specifi c antibodies against Ub (see Subheading 3.1). This approach requires a good antibody against the target working in IP; alternatively, one could express a tagged version of the protein, possibly at the endogenous level. Another approach consists in IP ubiquitinated proteins from total cell lysate followed by detection with the antibody against the protein of interest. This second method relies on the availability of specifi c and very effi cient antibodies against Ub (see Subheading 3.2). A more quantitative approach is the DELFIA assay (Perkin Elmer), an ELISA-based assay, which allows comparing more samples and conditions (see Subheading 3.3). Cross-validation with more than one approach is usually recommended in order to prove that your protein is modifi ed by ubiquitin. Here we will use the EGFR as model system but protocols can be easily modifi ed according to the protein of interest.
EGFR; ELISA; Endocytosis; Endogenous ubiquitination; Immunoprecipitation; Western blot
Settore MED/04 - Patologia Generale
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/443317
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