Lc10 hplc

Following CP purification, SDS-PAGE was performed on Biorad ReadyGels with a 4–20% Tris gradient. The gels were visualized by copper staining (0.5 M CuCl₂). High performance liquid chromatography (HPLC) was also used to determine the purity of CP and CP-PTX conjugate, using a LC10 HPLC (Shimadzu Scientific Instruments; Columbia, MD). For HPLC analysis of the CP, a Shodex OHPak KB-804 column (New York, NY) and isocratic flow of 1.0 mL min⁻¹of water: acetonitrile: formic acid [70:30:0.05] was used. For HPLC analysis of the CP-PTX conjugate, a Shodex OHPak SB-804 column (New York, NY) and isocratic flow of 0.5 mL min⁻¹ of PBS: acetonitrile [70:30] was used. The HPLC data was quantified by the integrated area under the peak at an absorbance of 228 nm, corresponding to the absorbance of PTX.

The hydrodynamic radii (R_h) of the ELP₁₆₀–srt–ABD–Dox and KEKE–ELP₁₆₀–srt–ABD–Dox conjugates were determined by DLS (DynaPro; Wyatt Technology, Santa Barbara, CA) at 25 °C using a single detector at 90°. Samples were prepared in sortase reaction buffer at 25 × 10⁻⁶ M, filtered through 0.22 μm Millex-GV filters (Sigma-Aldrich, St. Louis, MO), and measured using a DynaPro plate reader (Wyatt Technology, Santa Barbara, CA). The data were analyzed with a regularization fit of the autocorrelation function using a Rayleigh sphere model. The purity of the drug conjugates (KEKE–ELP₁₆₀–ABD–Dox and ABD–Dox) and efficiency of the sortase enzymatic reaction were assessed by size exclusion HPLC. Samples were injected into a LC10 HPLC (Shimadzu Scientific Instruments, Columbia, MD) with a Shodex OHPak SB-804 column (New York, NY) and PBS:acetonitrile (70:30 v/v) as the mobile phase at an isocratic flow rate of 0.3 mL min⁻¹. Eluting peaks were detected with a UV–vis detector set at 488 nm. Mass spectrometry analysis of the ABD–Dox conjugate was performed on a Bruker Autoflex Speed MALDI-TOFMS (Bruker Daltonics, Billerica, MA) using succinic acid matrix and porcine insulin as the internal standard.