Chapter title |
Plant Proteostasis
|
---|---|
Chapter number | 7 |
Book title |
Plant Proteostasis
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3759-2_7 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3757-8, 978-1-4939-3759-2
|
Authors |
Klecker, Maria, Dissmeyer, Nico, Maria Klecker, Nico Dissmeyer |
Editors |
L. Maria Lois, Rune Matthiesen |
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. |
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