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Peptide Arrays for Binding Studies of E3 Ubiquitin Ligases

Klecker et al., Methods in molecular biology (2016) - PMID: 27424747

Product(s) used in this publication:  PepStar™ Peptide Microarrays

Abstract:

The automated SPOT (synthetic peptide arrays on membrane support technique) synthesis technology has entrenched as a rapid and robust method to generate peptide libraries on cellulose membrane supports. The synthesis method is based on conventional Fmoc chemistry building up peptides with free N-terminal amino acids starting at their cellulose-coupled C-termini. Several hundreds of peptide sequences can be assembled with this technique on one membrane comprising a strong binding potential due to high local peptide concentrations. Peptide orientation on SPOT membranes qualifies this array type for assaying substrate specificities of N-recognins, the recognition elements of the N-end rule pathway of targeted protein degradation (NERD). Pioneer studies described binding capability of mammalian and yeast enzymes depending on a peptide's N-terminus. SPOT arrays have been successfully used to describe substrate specificity of N-recognins which are the recognition elements of the N-end rule pathway of targeted protein degradation (NERD). Here, we describe the implementation of SPOT binding assays with focus on the identification of N-recognin substrates, applicable also for plant NERD enzymes.

KEYWORDS:

N-end rule; Peptide library; Protein-protein interaction; ResPep SL; SPOT assay; Substrate screen; Ubiquitin ligase

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