UV–vis
absorption measurements were conducted on a Cary UV–vis 100
spectrophotometer (Agilent, USA). Protein concentration was determined
by UV–vis analysis on a Nanodrop 1000 instrument (Nanodrop,
USA) by monitoring absorbance at 280 nm. Electrospray LC/MS analysis
of proteins and their bioconjugates was performed using an Agilent
1200 series liquid chromatograph (Agilent Technologies, USA) that
was connected in-line with an Agilent 6224 time-of-flight (TOF) LC/MS
system equipped with a Turbospray ion source. Protein samples were
run with a Proswift RP-4H column (Dionex, USA). Protein mass reconstruction
was performed on the charge ladder with Mass Hunter software (Agilent,
USA). High-performance liquid chromatography (HPLC) was performed
on Agilent 1200 Series HPLC Systems (Agilent, USA). Sample analysis
for all HPLC experiments was achieved with an in-line diode array
detector and in-line fluorescence detector. SEC was performed using
a Polysep-GFC-P-5000 column (4.6 × 250 mm) (Phenomenex, USA)
at 1.0 mL/min using a mobile phase of 10 mM sodium phosphate buffer,
pH 7.2. DLS was performed on a Zetasizer Nano Series (Malvern Instruments,
UK). Measurements were taken in triplicate at protein concentrations
of 0.2–1.0 mg/mL in 10 mM sodium phosphate buffer, pH 7.2,
at 25 °C.
absorption measurements were conducted on a Cary UV–vis 100
spectrophotometer (Agilent, USA). Protein concentration was determined
by UV–vis analysis on a Nanodrop 1000 instrument (Nanodrop,
USA) by monitoring absorbance at 280 nm. Electrospray LC/MS analysis
of proteins and their bioconjugates was performed using an Agilent
1200 series liquid chromatograph (Agilent Technologies, USA) that
was connected in-line with an Agilent 6224 time-of-flight (TOF) LC/MS
system equipped with a Turbospray ion source. Protein samples were
run with a Proswift RP-4H column (Dionex, USA). Protein mass reconstruction
was performed on the charge ladder with Mass Hunter software (Agilent,
USA). High-performance liquid chromatography (HPLC) was performed
on Agilent 1200 Series HPLC Systems (Agilent, USA). Sample analysis
for all HPLC experiments was achieved with an in-line diode array
detector and in-line fluorescence detector. SEC was performed using
a Polysep-GFC-P-5000 column (4.6 × 250 mm) (Phenomenex, USA)
at 1.0 mL/min using a mobile phase of 10 mM sodium phosphate buffer,
pH 7.2. DLS was performed on a Zetasizer Nano Series (Malvern Instruments,
UK). Measurements were taken in triplicate at protein concentrations
of 0.2–1.0 mg/mL in 10 mM sodium phosphate buffer, pH 7.2,
at 25 °C.