Dbco nhs ester

Chemicals were purchased from Sigma-Aldrich and anhydrous organic solvents were Aldrich's Sure/Seal™. DBCO-NHS ester was purchased from Click Chemistry Tools. Peptides were custom synthesized by CPC Scientific (San Jose, C, USA) and oligonucleotides (PolyT₂₀) with a C12 amino modifier at its 5′-end by IDT (Integrated DNA Technologies). 1H and ¹³C NMR spectra were recorded at 400 MHz (^{1 (link)}H), 100 MHz (^{13 (link)}C), respectively. Chemical shifts are given in parts per million (ppm) on the delta scale (δ) and are referred to the solvent residual peak. HPLC analysis and purification were carried out in Agilent 1100 series equipped with a UV detector and a fraction collector. A Zorbax Eclipse Plus C18 column (4.6 × 150 mm, particle size 5 μm) from Agilent was used for the reversed phase HPLC. MALDI-TOF analysis was performed on Voyager-DE STR instrument.

O-GlcNAc mass tag was conducted according (Darabedian et al., 2018 (link)) with the following modification. HeLa cells were lysed with HEPES 10 mM pH 7.9, NaCl 300 mM, SDS 1% and sonicated for 5 s. lysates were clarified by centrifugation at 4°C for 15 min at full speed. Two-hundred micrograms of total protein was then subjected to GalNAz labeling using O-GlcNAc Click It kit (Invitrogen) according manufacturer’s instructions. Samples were then reduced and alkylated. Fifty micrograms of labeled protein was then incubated either with 10 mM of homemade 4.4 kDa DBCO-PEG or with equivalent volume of DMSO for 1 h at room temperature. Prior to western blot, samples were further purified by chloroform/methanol precipitation. Amino-dPEG₁₂-Tris(m-dPEG₂₄)₃ (Quanta Biodesign) was resuspended in methylene chloride under argon and incubated with 3 M excess of DBCO-NHS Ester (Click chemistry Tools) with gentle shaking overnight at room temperature. Reaction mixture was dried down and DBCO-dPEG₁₂-Tris(m-dPEG₂₄)₃ product was resuspended in water and filtered. Solution was vacuum dried in speedvac and resuspended at 50 mM in DMSO.