Infinite m200 fluorometer

Based on the hydrolysis of fluorogenic succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (suc-LLVY-AMC) peptides, the chymotrypsin-like activity of proteasomes was measured to determine their proteolytic activity. A suc-LLVY-AMC hydrolysis assay was carried out using purified proteasome and 12.5 μM suc-LLVY-AMC (Enzo Life Sciences) in assay buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mg/mL BSA, 1 mM ATP, and 1 mM DTT). The Ub-rho110 hydrolysis reaction was carried out using proteasomes or USP14 activated by vme-proteasomes, along with 20 nM or 100 nM Ub-rho110 in the presence or absence of 1 μg/mL (equivalent to 33 nM) RNA aptamers. RNA aptamers were incubated with proteasomes for 5 min before they were added to substrates. To examine the effects of RNA aptamers on USP14, RNA aptamers were incubated with USP14 for 5 min, then with vme-proteasomes for 5 min, before they were added to substrates. UCHL3 and USP47 were kindly provided by Eunice Eun-Kyeong Kim; USP5 was provided by Kyeong Kyu Kim. Proteasomal activity and deubiquitinating activity were monitored by measuring free AMC or rho110 fluorescence, respectively, in black 96-well plates using a TECAN infinite m200 fluorometer.

The ability of recombinant tau‐FL and tau‐AC proteins to accelerate microtubule assembly was evaluated by measuring DAPI fluorescence (excitation at 358 nm, emission at 461 nm) using a TECAN Infinite M200 fluorometer. In a final 100 µL volume reaction, 80 μm tubulin (T240, Cytoskeleton) was mixed with either tau‐FL or tau‐AC (molar ratio tau protein/tubulin = 1/10) in the PEM buffer (80 mm PIPES pH 6.9, 2 mm MgCl₂, 1 mM GTP, 10 µm DAPI, and 0.5 mm EGTA). Microtubules assembly was determined by monitoring DAPI fluorescence every 60 sec for 1 h at 37 °C. These experiments were performed in triplicate. For tubulin and tau co‐sedimentation, the samples were subjected to ultracentrifugation at 68 000× g for 20 min after the microtubule assembly assay. Pellet and supernatant fractions were analyzed separately using SDS‐PAGE.

The peptidase activity of TrCB1 forms processed as described above (0.45 μg TrCB1 per well, i.e., 70 nM) was assayed with two synthetic fluorogenic substrates (25 μM, Bachem): Z-Phe-Arg-AMC, (cathepsin L/B substrate) and Z-Arg-Arg-AMC (cathepsin B-selective substrate) in 50/100 mM citrate/phosphate buffer (CPB), 2 mM DTT, pH 3–8 (final volume 200 μl). The reactions were performed in 96-well black flat bottom plates (Nunc) using Infinite M200 fluorometer (TECAN) with excitation and emission wavelengths set to 360 and 465 nm, respectively. The release of AMC was measured at RT in a 30 min kinetic cycle at 2 min intervals.
An exopeptidase (peptidyl dipeptidase) activity assay using Bz-Gly-His-Leu as a substrate was performed with pepTrCB1 forms employing a modified protocol (Sajid et al., 2003 (link)). The pepTrCB1 forms (1 μg, 160 nM) were incubated for 15 min with the substrate in 50/100 mM CPB pH 4–5.5 containing 2 mM DTT (final volume 100 μl). Spontaneous reaction of the emerging free amino groups of His-Leu with fluorescamine (0.05 mg/ml) was monitored in a fluorometer set to 390/475 nm excitation/emission wavelengths during a 30 min kinetic cycle at 2 min intervals.