Maleimidocaproyl val cit pabc monomethyl auristatin e vcmmae

Conjugation of FGF1E-AviTag, FGF2_V and Affibody_HER2 with monomethyl auristatin E (MMAE) was performed with protein concentration of 0.5 mg/mL dissolved in PBS with 1 mM EDTA pH 7.4. The protein was added to maleimidocaproyl-Val-Cit-PABC-monomethyl auristatin E (vcMMAE) (MedChem Express, Monmouth Junction) at five-fold molar excess of MMAE over protein cysteine residues and incubated for 2 h at RT. After incubation, MMAE-FGF1-AviTag was purified by ion exchange chromatography using the HiTrap SP Sepharose FF column (GE Healthcare). The resin was washed with washing buffer containing 25 mM HEPES, 5% glycerol pH 7.4 to remove unconjugated MMAE and then MMAE-FGF1-AviTag was eluted with elution buffer containing 25 mM HEPES, 0.5 M NaCl, 10 mM Na₂SO₄, 5% glycerol pH 7.4. The purity and the identity of conjugate were confirmed by SDS-PAGE.

The chromatographic columns
HiTrap MabSelect
SuRe and HiTrap Desalting with Sephadex G-25 resin were obtained from
GE Healthcare (UK). Reagents for microscopy CellLight Early Endosomes-RFP,
BacMam 2.0 (#C10587), Zenon Alexa Fluor 488 Human IgG Labeling Kit
(#Z25402), and NucBlue Live ReadyProbes Reagent (#R37605) were purchased
from Thermo Fisher Scientific (Waltham, MA). Conjugation reagents
tris(2-carboxyethyl) phosphine (TCEP) pH 7.0 (#646547) was obtained
from Merck (Darmstadt, Germany), maleimidocaproyl-Val-Cit-PABC-monomethyl
auristatin E (vcMMAE) (#HY-15575, MedChem Express).