Alexa 488 c5 maleimide

Prior to labeling, a G25 column (GE Healthcare) was used to exchange Arc-cys-st11-ssrA into buffer E (50 mM Tris, pH 7.5, 300 mM NaCl, 1 mM EDTA) or to exchange Arc-cys-st11-sul20 into buffer F (50 mM Tris, pH 7.5, 300 mM NaCl, 5% (v/v) glycerol, 0.032% (v/v) Igepal CA-630, 1 mM EDTA). Alexa488-C5-maleimide (Thermo Fisher) was added to the buffer-exchanged samples in a 4:1 molar ratio of fluorophore to protein (in monomer equivalents), and the reaction was incubated in the dark for 2 h at room temperature. The reaction was quenched by addition of 5 mM DTT, and the labeled product was sequentially chromatographed on a PD-10 desalting column (GE Healthcare) and a HiLoad Superdex 75 16/60 column, both equilibrated in buffer D. Appropriate Superdex-75 fractions were pooled, concentrated, and the final concentration was determined by the Bradford assay (Bio-Rad) using unlabeled Arc-cys variants as standards.

Conjugating Alexa488 C5 maleimide (Invitrogen #A10254) to the N-terminus cysteine of pHLIP was achieved by dissolving pHLIP in DMF with 50 mM HEPES, pH 7.2, followed by the addition of 1 molar equivalent of Alexa488 C5 maleimide. The solution was flushed with nitrogen and mixed at room temperature for 4 hours. pHLIP-MMAF was prepared by conjugating Py-ds-Prp-MMAF (Levena Biopharma) to pHLIP with a C-terminus cysteine residue utilizing the same procedure. The desired pHLIP conjugates were isolated using the same techniques described for the pHLIP peptides. The purity of the pHLIP conjugates were determined by RP-HPLC, and their identity was confirmed by MALDI-TOF MS. Alexa488-pHLIP: purity >99%; calculated (M+H+) = 4874, found (M+H+) = 4874. pHLIP-MMAF: purity >98%; calculated (M+H+) = 5030, found (M+H+) = 5030. The Alexa488-pHLIP conjugate was quantified at 493 nm by UV/Vis absorbance spectroscopy using the molar absorption coefficient of Alexa488 C5 maleimide (72,000 M⁻¹⋅cm⁻¹). The pHLIP-MMAF conjugate was quantified at 280 nm using the molar absorption coefficient of pHLIP (13940 M⁻¹⋅cm⁻¹). The conjugates were then lyophilized in 10⁻⁸ mole aliquots.

Purified full length MBP-BCL10, BCL10 R58Q and BCL10 E140X were mixed with 5-fold molar excess of Alexa- 488-C5-maleimide (Invitrogen) and incubated at 4 °C temperature for O/N. Gel filtration chromatography (Superdex 200, GE Healthcare) was used to remove free dyes. Fluorescence polarization assay was performed at 18 °C in buffer containing 20 mM Tris at pH 7.5, 150 mM NaCl, and 0.5 mM TCEP and in 20 μl volume. 3 μM of labeled MBP- BCL10 were cleaved with 3C in the presence of increasing amount of MALT1. The fluorescence quenching was measured right after 3C addition for 2 h by using NEO plate reader (Biotek) using excitation/emission wavelengths of 495 nM/519 nM.