Dbco peg4 nhs

The primer–template
hybrid for conjugation to detector antibody was prepared by first
annealing a 5′ azide-modified primer (36.1 μM) and a
linear methylated AluI D6 template (37.9 μM) in NEBNext Quick
Ligation Buffer (New England Biolabs). The mixture was heated at 95
°C for 2 min and allowed to slowly cool to room temperature over
1.5 h. The template was then ligated by adding T4 DNA ligase and incubating
at room temperature for 3 h. After ligation, a 7K MWCO Zeba spin desalting
column (Thermo Fisher Scientific) was used to buffer exchange the
primer–template pair into phosphate buffered saline (PBS) with
1 mM EDTA. The detector antibody (AF6274, R&D Systems) was reconstituted
to 1 mg/mL in PBS and incubated with a 20-fold molar excess of dibenzocyclooctyne-PEG4-N-hydroxysuccinimidyl ester (DBCO-PEG4-NHS, MilliporeSigma)
at room temperature for 30 min before purification with a 10K Amicon
Ultra-0.5 mL centrifugal filter in PBS with 1 mM EDTA. The DBCO-modified
detector antibody was then mixed with a 2-fold molar excess of the
ligated primer-template and incubated at 4 °C overnight. The
antibody–DNA conjugate was aliquoted and stored at −80
°C in PBS with 5 mM EDTA, 0.1% BSA, and 0.02% sodium azide.

Phosphoramidite reagents used for the synthesis of FNA were purchased from Glen Research (VA, United States): spacer 18 phosphoramidite (10–1918), DBCO-dT-CE phosphoramidite (10–1539), Thiol-Modifier C6 S-S (10–1936), biotin CPG (20–2993). N-terminus azide modified Aβ42 [K(N3)-Aβ42] peptide was purchased from GenScript Biotech (NJ, United States). N-(g-Maleimidobutyryloxysuccinimide ester) GMBS was purchased from Pierce Biotechnology (Grand Island, NY). Streptavidin was obtained from Sigma-Aldrich (MO, United States) and Tris-(2-Carboxyethyl) phosphine-HCl (TCEP-HCl) was purchased from Hampton Research Inc. (CA, United States). 1-(3-aminopropyl) silatrane (APS) was synthesized as previously described (Shlyakhtenko et al., 2013 (link)). β-mercaptoethanol, NHS-PEG₄-DBCO and all other reagents or solvents were purchased from Sigma–Aldrich (MO, United States).