Typhoon fla 9500 gel scanner

Manufactured by GE Healthcare

The Typhoon FLA 9500 is a gel scanner designed for high-resolution imaging of fluorescent and chemiluminescent gels and blots. It utilizes a laser-based detection system to capture images with exceptional sensitivity and resolution.

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Lab products found in correlation

Proteinase k, by Thermo Fisher Scientific (2 mentions) Mes buffer, by Thermo Fisher Scientific (1 mentions) Nupage 4 12 bis tris sds gel, by Thermo Fisher Scientific (1 mentions)

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4 protocols using typhoon fla 9500 gel scanner

Denaturing PAGE Gel Electrophoresis Protocol

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For these experiments, we used 10% PAGE TBE-Urea precast Gels (Fisher Scientific). For each sample, roughly 2 pmol oligo was dissolved in 1x loading buffer, the latter comprising 48% formamide (v/v), 10 mM EDTA, and 1 mg/mL bromophenol blue. The mixture was then denatured by incubating at 95 °C for 10 min, and then loaded into the gel. Electrophoresis was run for 45 min at 60 °C at 110 V. The gel was then imaged using a Typhoon FLA 9500 gel scanner (GE Healthcare). Quantitation of gel band intensities was performed using accompanying software. Uncropped gel images are available in the Supplementary Information.

Pinto A., Chen S.X, & Zhang D.Y. (2018). Simultaneous and stoichiometric purification of hundreds of oligonucleotides. Nature Communications, 9, 2467.

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Quantitative Ubiquitin Cleavage Assay

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All cleavage reactions were done in reaction buffer 25 mM HEPES pH 7.5, 150 mM NaCl, 5 mM DTT. NCPs ubiquitinated on either one of the H2A ubiquitination sites with ^TAMRAUb were used and reaction was started by addition of USP48. Concentrations are indicated in the figures. Samples were taken at the indicated time points and reaction was stopped by addition of SDS-loading dye. Samples were separated on a NuPage 4–12 % Bis-TRIS SDS gel in MES buffer (Thermo Fisher) and the fluorescence signal was read out on a Typhoon FLA-9500 gel scanner (GE Healthcare). Quantification of individual bands was achieved by measuring the fluorescence intensity and relating it to the total intensity of each lane at a known concentration of dye used in the respective assay. This way each band represents a fraction of the concentration of dye used in the experiment. To convert to molar concentration of ubiquitinated histones the dye concentration was divided by the number of labeled ubiquitins present on the respective histone species (H2Aub3, H2Aub2, and H2Aub1).

Uckelmann M., Densham R.M., Baas R., Winterwerp H.H., Fish A., Sixma T.K, & Morris J.R. (2018). USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A. Nature Communications, 9, 229.

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Visualizing RNP-DNA Interactions with Fluorescence

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ATTO647N-labeled target DNA was generated with 20 rounds of PCR using Q5 DNA polymerase (NEB) and oligonucleotides 365, 460 and 371. The DNA was diluted in series from 512 nM to 4 nM in reaction buffer (20 mM HEPES, pH 7.5, 150 mM KCl, 10 mM MgCl₂, 2 mM DTT). RNP complexes were formed by mixing protein and RNA (256 nM:384 nM) and incubating for 30 min at room temperature in the same buffer conditions. Equal volumes of RNP and ATTO647N-labeled matched DNA dilutions were combined and then incubated for 30 min at room temperature. The reaction was halted by the addition of a stop solution (40 mM EDTA and 50 U proteinase K (Thermo Fisher Scientific)), and a 30-min incubation at 37 °C removed RNPs from their DNA substrates. All samples were run in a 10% polyacrylamide native gel and then imaged using a Typhoon FLA9500 gel scanner (GE Healthcare).

Jones SK J.r., Hawkins J.A., Johnson N.V., Jung C., Hu K., Rybarski J.R., Chen J.S., Doudna J.A., Press W.H, & Finkelstein I.J. (2020). Massively parallel kinetic profiling of natural and engineered CRISPR nucleases. Nature biotechnology, 39(1), 84-93.

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PCNA-Ubiquitin Cleavage Kinetics Assay

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PCNA‐Ub^TAMRA cleavage assays were performed in a reaction buffer composed of 20 mM HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 0.25 mM ATP, 2 mM DTT. The cleavage reaction was started by addition of USP1 (±UAF1) followed by incubation at room temperature for the specified time course. The concentration of DUB used for the kinetic modelling experiments was 1, 0.5, 0.25 µM for USP1 and 20, 10, 5 nM for USP1‐UAF1. All the mutants tested for activity on PCNA‐Ub^TAMRA were in complex with UAF1, and they were run in parallel with wild‐type USP1 at a concentration of 25 nM. Samples were collected at the indicated time points, and the reaction was stopped by adding SDS loading buffer. Samples were loaded on NuPAGE 4–12% Bis‐Tris SDS gel (Invitrogen) and separated by running them at 180 V for 30 min. The TAMRA fluorescence signal was visualized using a Typhoon FLA‐9500 gel scanner (GE Healthcare), and the concentration of PCNA‐Ub^TAMRA and ubiquitin was quantified by comparing the TAMRA fluorescence of the individual bands with a calibration curve of TAMRA fluorescence in the same experimental setting.

Dharadhar S., van Dijk W.J., Scheffers S., Fish A, & Sixma T.K. (2021). Insert L1 is a central hub for allosteric regulation of USP1 activity. EMBO Reports, 22(4), e51749.

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