Controlled immobilization of His-tagged proteins for protein-ligand interaction experiments using Ni²âº-NTA layer on glass surfaces.
Clin Hemorheol Microcirc
; 61(3): 523-39, 2015.
Article
de En
| MEDLINE
| ID: mdl-26444589
Gold surfaces functionalized with nickel-nitrilotriacetic acid (Ni²âº-NTA) as self-assembled monolayers (SAM) to immobilize histidine (His)-tagged biomolecules are broadly reported in the literature. However, the increasing demand of using microfluidic systems and biosensors takes more and more advantage on silicon technology which provides dedicated glass surfaces and substantially allows cost and resource savings. Here we present a novel method for the controlled oriented immobilization of His-tagged proteins on glass surfaces functionalized with a Ni²âº-NTA layer. Exemplarily, the protein pattern morphology after immobilization on the Ni²âº-NTA layer of His6-tagged soluble receptor for advanced glycation endproducts (sRAGE) was investigated and compared to non-oriented immobilization of sRAGE on amino SAM by using scanning electron microscopy (SEM). Moreover, we demonstrated interaction of immobilized sRAGE with three structurally different ligands, S100A12, S100A4, and glycated low density lipoproteins (glycLDL), by means of peak-force tapping atomic force microscopy (PF-AFM). We showed a clear discrimination of different protein-ligand orientations by differential height measurements.
Mots clés
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
Oligopeptides
/
Composés organométalliques
/
Histidine
/
Acide nitrilo-triacétique
Langue:
En
Journal:
Clin Hemorheol Microcirc
Sujet du journal:
ANGIOLOGIA
/
HEMATOLOGIA
Année:
2015
Type de document:
Article
Pays d'affiliation:
Allemagne
Pays de publication:
Pays-Bas