Hitrap sp sepharose ff column

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 GST-tagged p300 Core protein was purified through affinity purification using the GST HiTrap column (Sigma) followed by ion-exchange on HiTrap SP Sepharose FF column (GE Healthcare). The protein was washed with buffer containing 20 mM HEPES pH 7.0, 50 mM KCl, 0.5 mM TCEP, and 10% glycerol, and eluted with the same buffer containing 300 mM KCl. Fractions of peak center were collected and used for crosslink. His-tagged full-length p53 was purified by affinity purification using Ni-NTA resins followed by gel filtration on HiLoad 16/600 Superdex 200 column (GE Healthcare). The column was equilibrated with 20 mM HEPES, pH8.0, 300 mM KCl, 0.5 mM TCEP and 10% glycerol, and the p53 proteins at peak center were collected. For p53 competition, 115 μg of the p300 Core protein was mixed with 207 μg of p53 (at the molecular ratio of 1:4) and incubated at RT for 10 min before adding 1 mM DSSO for crosslinking. Reactions were quenched by adding 20 mM NH₄HCO₃. After reduction by 2 mM DTT and alkylation by 4 mM chloroacetamide, samples were digested with 2% Trypsin (Promega) at 37° for 20 h and quenched by adding 0.1% formic acid. After C18 tips desalting, peptides were analyzed by mass spectrometry at the UCI High-end Mass Spectrometry Facility. Data were processed by ProteoWizard MSConvert (v. 3.0.10738) and Protein Prospector (v.5.19.1).