Phosphate → HCID 9836845, compound

Participants

Reaction Steps

A 5 mg/mL solution of ovalbumin in PBS (100 mM phosphate, 150 mM NaCl, pH 7.4) and 2 mM EDTA (200 μL; 1 mg protein) was prepared. A solution of succinimidyl 6-hydrazinonicotinate acetone hydrazone (SANH)(EXAMPLE 2)(2 mg) in DMF (50 μL) is prepared. To the protein solution was added the SANH/DMF solution (15 eq.). The reaction mixture was incubated at room temperature for 4 hrs. The modified protein was isolated and buffer exchanged by placing the reaction mixture in a 30K ultra-free centrifugation device and washing three times with 0.1 M MES, 0.9% NaCl, pH 4.7 (3×400 μL). The purified protein was quantified for protein concentration (BCA assay; Pierce Chemical Co., Rockford, Ill.) and for hydrazine modification level by incubation of an aliquot of protein in a 0.5 mM 4-nitrobenzaldehyde in 0.1 M MES, 0.90% NaCl, pH 4.7 and measuring the absorbance at 360 nm (molar extinction coefficient 22,000).

Provenance & Evidence