Chapter title |
Oriented Peptide Immobilization on Microspheres.
|
---|---|
Chapter number | 14 |
Book title |
Peptide Microarrays
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3037-1_14 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3036-4, 978-1-4939-3037-1
|
Authors |
Lisa C. Shriver-Lake, George P. Anderson, Chris R. Taitt, Shriver-Lake, Lisa C., Anderson, George P., Taitt, Chris R. |
Editors |
Marina Cretich, Marcella Chiari |
Abstract |
Reproducible immobilization of peptides and proteins on microsphere surfaces is a critical factor for optimal sensitivity and selectivity in bead-based assays. However, peptides with unusually large numbers of lysine residues-whose amines are targeted in the most common microsphere immobilization chemistries-may be particularly challenging to use in bead-based arrays, as they may lose activity through multipoint attachments and incorrect presentation. For this reason, it is imperative to achieve site-directed attachment chemistry, such that a single site of attachment provides reproducibly oriented peptides on the microsphere surface. This can be achieved by inserting a unique targetable residue, such as a cysteine. Here, we present methods for attaching cysteine-containing peptides to standard carboxy-functionalized microsphere surfaces using thiol- rather than amine-directed chemistries. We show that the presence of a cationic detergent (CTAB) and a "passivating" agent such as β-mercaptoethanol facilitates improved bead recovery after peptide immobilization and may enhance functionality of the attached peptides. |
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