Infinite m1000 pro fluorometer

The DNA ligation products for site-directed or random mutagenesis were transformed into DH5α cells lacking adenylate cyclase gene CyaA(DH5α-ΔCyaA). After overnight growth at 34°C, the colonies with different fluorescence intensities on the LB agar plates were screened by eye in a BlueView Transilluminator (Vernier) with the 400–500 nm excitation light and a yellow acrylic long-pass filter, or by fluorescence imaging in a home-made imaging system with 480/20 nm excitation and 520/20 nm emission filters. To quantitatively compare the brightness of selected variants, bacterial patches on the agar plates cultured overnight at 34°C were imaged in the home-made system mentioned above and analyzed by ImageJ software (National Institutes of Health). The fluorescence change (ΔF/F₀) of the cAMP sensor mutants were tested as previously described (Wang et al., 2022 (link)). Briefly, selected bacterial colonies were patched on LB agar plate and grew at 25°C for 3 days. Then the bacteria from each patch were collected, suspended in HEPES buffer (pH = 7.15) and lysed by sonication. The clear lysates were then used for measuring the ΔF/F₀ in response to 500 μM cAMP with an Infinite M1000 PRO fluorometer (Tecan).

The in vitro DUB enzymatic assays using Ub-AMC as substrate was performed as described previously⁵⁵ . Briefly, 1 μL Ub-AMC (Boston Biochem) was performed in 50 μL reaction buffer (20 mM HEPES-KOH pH 7.8, 20 mM NaCl, 0.1 mg/mL ovalbumin, 0.5 mM EDTA, and 10 mM DTT) at room temperature, and then fluorescence at emission 460 nm and excitation 380 nm was monitored in an Infinite M1000 PRO Fluorometer (TECAN).

Immunoproteasome (β5i and β1i) activity assay was performed as previously described. Briefly, 20 mg of heart tissue samples were homogenized in immunoproteasome buffer (50 mM Tris, 5 mM MgCl₂, 20 mM potassium chloride (KCl), 1 mM DTT (freshly added), pH 7.5) with a hand-held Potter-Elvehjem homogenizer. The homogenates were centrifuged at 12,000× g for 15 min at 4 °C and supernatant collected. Next, 20 μg of protein were incubated with immunoproteasome buffer, and specific immunoproteasome inhibitor or an equivalent volume of DMSO at RT for 20 min protected from direct light exposure. The specific inhibitors for β5i 20 µM ONX-0914 (Abmole Bioscience Inc., Houston, TX, USA, Cat. No. M2071) and β1i 50 µM bortezomib were used to control for non-immunoproteasome-mediated cleavage of substrates and evaluate the specificity of the assay. Then, 25 µM fluorogenic substrates Ac-ANW-2R110 (AAT Bioquest, Inc., Pleasanton, CA, USA) for β5i and Ac-PAL-2R110 (AAT Bioquest, Inc., Pleasanton, CA, USA) for β1i were used to initiate the reaction. The fluorescence intensity was measured every 5 min for 60 min at an excitation of 498 nm and an emission of 520 nm at 37 °C in a Tecan Infinite M1000 Pro fluorometer.

For AICD peptide uptake analysis in cell lysates MEF APPΔCT15 were incubated with FITC-AICD as decribed above. Under light exclusion cells were washed 5 times with PBS to remove attached FITC-AICD peptides and lysed in lysis buffer (150 mM NaCl, 50 mM Tris/HCl pH 7.4, 2 mM EDTA, 0.1% NP-40, 0.1% Triton-X 100). Lysates were adjusted to equal protein content and dispensed on black 96-well plates (Corning, Lowell, MA, USA). Fluorescence signal of FITC-AICD was determined at an excitation wavelength of 495 ± 10 nm and an emission wavelength of 521 ± 10 nm using an Infinite M1000Pro Fluorometer (Tecan, Crailsheim, Germany).

The 26 S proteasome assay was carried out as described previously [31 (link)–33 (link)]. A total volume of 100 μl was incubated in 96 well plates containing 100 μM ATP in 26S buffer and 20 μg of protein supernatants. Assays were initiated by addition of fluorescently labeled substrate: succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC), Boc-Leu-Ser-Thr-Arg-AMC (Boc-LSTR-AMC, Bachem), and Z-Leu-Leu-Glu-AMC (Z-LLE-AMC) for chymotrypsin- (β5), trypsin- (β2) and caspase- (β1) like activity measurements, respectively. The final concentration of substrate in each assay was 100 μM.
These substrates are cleaved by the proteasome, releasing free 7-amino-4-methylcoumarin (AMC) which was then measured spectrofluorometrically using an Infinite M1000 PRO fluorometer (Tecan, Männedorf, Switzerland) at an excitation wavelength of 390 nm and an emission wavelength of 460 nm. Fluorescence was measured at 15-min intervals for 2 h. Each assay was conducted in the absence and presence of the specific proteasomal inhibitor bortezomib (LC labs, Woburn, MA), 10 μM for β5 chymotrypsin-like activity, and 100 μM for β2 trypsin-like activity, and β1 caspase-like activity. All assays were assessed for linearity, and the results of either of the two time-points with the highest proteasome activity in the linear range (i.e., 75 min and 90 min) were used for analysis.

tRNA binding was measured by fluorescence anisotropy using wild-type and Trm10-KRR variant proteins and 5’−6-carboxyfluorescein-labeled tRNA^Gly-GCC transcripts, prepared, and measured as previously described (17 (link)). Reactions containing varied concentration of enzyme (50–1000 nM) and 15 nM fluorescently-labeled tRNA were incubated for 30 minutes at room temperature prior to measuring anisotropy using an Infinite M1000 PRO fluorometer (Tecan). Anisotropy was measured and plotted as a function of concentration of each Trm10 enzyme. Control binding reactions contained identical concentrations of bovine serum albumin (BSA) and fluorescently-labeled tRNA. The data were fit to equation 1 (a modified Hill equation) using Kaleidagraph (Synergy Software) to yield the observed ${\mathrm{K}}_{\mathrm{D}}$ , the minimum anisotropy ( $\mathrm{F}{\mathrm{A}}_{\mathrm{m}\mathrm{i}\mathrm{n}}$ ), maximum anisotropy ( $\mathrm{F}{\mathrm{A}}_{\mathrm{m}\mathrm{a}\mathrm{x}}$ ) and Hill coefficient (n) for each enzyme.