Oriented Peptide Immobilization on Microspheres.
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.
US Naval Research Laboratory, Code 6900, 4555 Overlook Avenue, SW, Washington, DC, 20375, USA.
Lisa C
LC
Shriver-Lake
US Naval Research Laboratory, Code 6900, 4555 Overlook Avenue, SW, Washington, DC, 20375, USA.
George P
GP
Anderson
US Naval Research Laboratory, Code 6900, 4555 Overlook Avenue, SW, Washington, DC, 20375, USA. chris.taitt@nrl.navy.mil.
Chris R
CR
Taitt
D000588:Amines / Q000737:chemistry; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry*; D002234:Carbodiimides / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D055767:Immobilized Proteins / Q000737:chemistry*; D008301:Maleimides / Q000737:chemistry; D008863:Microspheres; D011092:Polyethylene Glycols / Q000737:chemistry; D040081:Protein Array Analysis; D017433:Protein Structure, Secondary; D013388:Succinimides / Q000737:chemistry; D013499:Surface Properties
D000588:Amines; D023181:Antimicrobial Cationic Peptides; D002234:Carbodiimides; D055767:Immobilized Proteins; D008301:Maleimides; D013388:Succinimides; C074139:1-ethyl-3-(3-dimethylaminoethyl)carbodiimide; C043592:maleimide; D011092:Polyethylene Glycols; D003545:Cysteine; C001426:N-hydroxysuccinimide
Antimicrobial peptide; Bead-based flow cytometry; Bioimmobilization; Luminex