Fluorescence biotin quantitation kit

Amino-dextran was synthesized as previously reported (Nakamura et al., 2010 (link)). The synthesis and chemical characterization of dextran conjugates were described in details in our previous paper (Morimoto et. al., Bioconjugate Chem. 2014). In short, dextran (500 mg, average molecular weight 35,000–45,000, from Leuconostoc mesenteroids, Sigma) was dissolved in 50 mL of anhydrous DMF and treated with 126 mg of N,N′-carbonyldiimidazole and 250 µL of ethylenediamine. Aminodextran was treated with biotin-NHS ester in DMSO at 4 °C for overnight to conjugate biotin. COPA or peptide ligands were conjugated to the biotinylated-dextran polymer through N-(α-Maleimidoacetoxy) succinimide ester (AMAS, from Thermo scientific) in DMSO for two hours in the dark. The unreacted reactants were removed by filtering through Amicon Ultra 4 mL centrifugal filter unit (10,000 NMWL, from Millipore). The ligand-dextran conjugate in the filter unit was recovered in 1×PBS according to the manufacturer's protocol. The concentration of biotin in the solution was quantitatively determined using the Fluorescence Biotin Quantitation Kit (Thermo Scientific). The procedure to estimate the number of ligands conjugated to dextran polymer is described in details in Supplemental experimental procedures.

Unincorporated free biotin and biotin incorporated to proteins were measured using a Fluorescence Biotin Quantitation Kit (Thermo) and a fluorescence plate reader (Synergy Mx Microplate Reader, Biotek) by measuring fluorescent excitation/emission at 495/520 according to manufacturer’s instructions. Dissected samples from retina and LGN from 6 animals were weighed and pooled. Protein extracts were generated by homogenizing and sonicating 122 mg of wet tissue from LGN or retina in 1ml of 1 mM EDTA, 50 mM Tris, pH7.5 (TE) buffer and centrifuged at 10,000 g for 15 min at 4°C to remove nuclei and cell debris. The lysates were mixed with 4 mL of cold acetone, incubated at −20°C overnight and centrifuged for 1 h at 4500 g in a swinging bucket rotor to precipitate proteins. Supernatants were collected and evaporated to a volume of ~20 μL in a SpeedVac concentrator (Thermo Scientific) and protein pellets were solubilized in RIPA buffer. As a standard, we used a series of biocytin dilutions (from 0.5 to 10 pmol/μl) according to the manufacturer’s instructions.