Typhoon gel scanner

In a typical antibody-photosensitizer conjugate synthesis, 1 mg of CD44 mAb was first dispersed in 1 ml of 1X PBS and then added with 100 μl of K₂HPO₄ buffer (pH = 9.0) and 159.2 μg of IR700 (81.6 nmol, 1 mM in DMSO). The mixture was kept at 4 °C for overnight, and then placed in ultra-centrifugal filter units (Millipore Amicon Ultra-0.5, 10 kDa, Billerica, MA) to remove the unbound IR700 molecules. The IR700-conjugated anti-CD44 monoclonal antibody obtained was abbreviated as CD44-IR700. Similarly, we obtained mouse IgG conjugated with IR700 as a non-target control, which was abbreviated as IgG-IR700. The concentration of antibody and dye/protein ratio was determined spectroscopically by measuring the absorbance of the conjugate at 280 nm and 689 nm. The extinction coefficients were 210,000 M⁻¹cm⁻¹ for CD44 mAb at 280 nm, and 165,000 M⁻¹cm⁻¹ for IR700 at 689 nm. The correction factor of IR700 at 280 nm was set to be 0.095. The purity of CD44-IR700 was checked with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) performed on a 4–20% gradient gel (Mini-PROTEAN TGX precast gels, BIO-RAD, Hercules, CA). Fluorescence from the gel was obtained from a Typhoon gel scanner (GE Healthcare Bio-Sciences, Piscataway, NJ), and the protein bands on the gel were stained with GelCode^® blue stain reagent (Thermo Fisher Scientific, Grand Islands, NY).

Samples were prepared for alkaline gel electrophoresis in 6x Purple Loading Dye (NEB). Agarose gels (0.4%) were prepared in alkaline buffer (50 mM NaOH, 10 mM EDTA) and electrophoresis was performed in the same buffer at 1 V cm^-1 and 4 °C for 16 hours with buffer circulation. Gels were neutralized in 50 mM Tris pH 7.5 buffer, stained with SYBR Gold (Thermo Fisher) and visualized on a Typhoon gel scanner (GE Healthcare).