Amersham typhoon scanner

The strand separation assays (10 μl volume) were carried out in SS buffer (25 mM HEPES pH 7.5, 60 mM NaCl, 0.1 μg/μl BSA, 1 mM MgCl2, 1 mM ATP, 10 mM creatine phosphate, 15 μg/ml creatine kinase) containing indicated fluorescently labelled DNA substrate (5 nM). The reactions were started by addition of increasing amounts of Mer3 protein (10, 20, 40 and 80 nM). After the incubation for 30 min at 30°C the reactions were stopped with 0.5 mg/ml proteinase K and 0.1% SDS, and incubated for 5 min at 37°C. The samples were then mixed with 2 μl of the gel loading buffer (60% glycerol, 10 mM Tris–HCl, pH 7.4, 60 mM EDTA) and separated on 10% (w/v) native polyacrylamide gel in 1× TBE buffer at a constant voltage of 110 V for 1 h at 4°C. The gels were scanned using Amersham Typhoon scanner (Cytiva) and quantified by ImageQuant TL software (Cytiva).

PUREfrex (GeneFrontier) with Cy5-labeled tRNA^fMet was used to express RNA templates produced by CUGA7 in vitro transcription kit (Nippon Gene) in the presence or the absence of purified IbpAs or the mutants (1 μM). The reaction mixtures were incubated at 37 °C for 2 h, then mixed with the same volume of 2× SDS sample buffer (0.5 M Tris–HCl pH 6.8, 10% SDS, 10% (w/v) sucrose, 0.01% (w/v) bromophenol blue, and 10% (w/v) 2-mercaptoethanol), and boiled at 95 °C for 5 min. The mixtures were separated by SDS-PAGE, and visualized by an Amersham Typhoon scanner (Cytiva), and finally quantified by Multi gauge software (Fujifilm).

The binding reactions (10 μl volume) were carried out in EMSA buffer (25 mM HEPES pH 7.5, 0.1 μg/μl BSA, 60 mM NaCl) containing indicated fluorescently labelled DNA substrate (10 nM). The reactions were started by addition of increasing amounts of Mer3 protein (37.5, 75, 150 and 300 nM) or Mlh1–Mlh2 complex (20, 40, 75 and 150 nM) and incubated for 20 min at 30°C. After the addition of 2 μl of the gel loading buffer (60% glycerol, 10 mM Tris–HCl, pH 7.4, 60 mM EDTA, 0.15% Orange G), the reaction mixtures were resolved in 0.8% agarose gel in 1× TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA). The gels were scanned using Amersham Typhoon scanner (Cytiva) and quantified by ImageQuant TL software (Cytiva).

For the hydroxyl-radical footprinting experiments (46–48 (link)), the fluorescently labeled nucleosome (0.6 µm) was mixed with either p53 (2.4 µm), p53 DBD (3.6 µm), or p53 CTR (3.6 µm), and was incubated at 25ºC for 30 min. The buffer was exchanged into the reaction buffer, containing 5 mm Tris-HCl (pH 7.5), 5 mm NaCl, and 0.25 mm EDTA (pH 8.0), by four consecutive rounds of dilution and filtration using an Amicon Ultra-0.5 filter unit (MWCO 30 kDa). For the hydroxyl radical reaction, a 2.5 µL aliquot of 4 mm FeAmSO₄/8 mm EDTA, 0.1 m sodium ascorbate, and 0.6% v/v H₂O₂ was simultaneously mixed with 50 µL of the sample, and then incubated for 2 min at room temperature. The reactions were terminated by adding a 5 μL aliquot of 100 mm thiourea and a 10 μL aliquot of 3 m sodium acetate (pH 5.2). The resulting DNA samples were purified by deproteinization and phenol/chloroform/isoamyl alcohol extraction, followed by ethanol precipitation. The DNA samples were denatured by Hi-Di^™ formamide (Thermo Fisher Scientific) and separated by 8% Urea-PAGE in 1×TBE buffer. The Cy5 and 6FAM signals were detected with the Amersham Typhoon scanner (Cytiva). The gel images were analyzed with the ImageJ software (49 (link)).

Synthetic vRNA loop (‘v51_mut_S’) (5′-pAGU AGA AAC AAG GGU GUA UUU UCC CCU CUU UUU GUU UCC CCU GCU UUU GCU -3′) (IDT) was used for all in vitro replication activity assays. For all de novo replication activity assays, 2.4 μM FluPol Zhejiang-H7N9 (WT or 4 M) were mixed with (i) 0.8 μM v51_mut_S and/or (ii) 8 µM ANP32A and/or (iii) 16 µM pS5 CTD(6mer) (respective molar ratio: 3 FluPols: 1 template: 10 ANP32: 20 pS5 CTD). Reactions were launched at 30 °C for 4 h by adding ATP/GTP/CTP/UTP (AGCU) or only ATP/GTP/CTP (AGC), α-32P ATP (PerkinElmer) and MgCl₂ in a final assay buffer containing 50 mM HEPES pH 8, 150 mM NaCl, 2 mM TCEP, 100 μM/NTP, 1 mM MgCl₂, 0.05 μCi/μl α-32P ATP. Reactions were stopped by adding 2X RNA loading dye, heating 5 min at 95 °C and immediately loaded on a 20% TBE-7M urea-polyacrylamide gel. Each gel was exposed on a storage phosphor screen and read with an Amersham Typhoon scanner (Cytiva). For each gel the decade markers system (Ambion) was used.

To verify whether Sec:msfGFP and Tat:msfGFP were secreted and folded correctly, and to confirm whether Coa:msfGFP was secreted as an intact, functionally fluorescent protein, we separated the supernatant on a native PAGE gel. Cultures that expressed either Coa:msfGFP, Tat:msfGFP, msfGFP, an empty vector were grown to the exponential phase in mM9, and the chromosome-integrated Coa:msfGFP cultures were grown to the stationary phase in BHI, after which the supernatant was collected and either stored at –80°C or used right away. The supernatants were mixed 1:1 with a native sample buffer (1610738, Bio-Rad) and separated on a 4–15% precast polyacrylamide protein gel (Mini-PROTEAN TGX, 4561086, Bio-Rad). Fluorescence was detected in the Amersham Typhoon Scanner (29187191, Cytiva) with a 488 nm excitation and 510 nm emission. A His-tagged GFP (14-392, Sigma-Aldrich) was also loaded on the gel and used as a positive control for GFP fluorescence as well as a size marker.

The binding reactions (10 μL volume) were carried out in EMSA buffer (25 mM HEPES pH 7.5, 0.1 μg/μL BSA, 60 mM NaCl) containing indicated fluorescently labelled DNA substrate (10 nM). The reactions were started by addition of increasing amounts of SU_C protein complexes (36.25, 72.5, 108.75, 145, 217.5, 290, 435, 580, 870, 1160 and 1740 nM) and incubated for 20 min at 30°C. After the addition of 2 μL of the gel loading buffer (60% glycerol, 10 mM Tris–HCl, pH 7.4, 60 mM EDTA, 0.15% Orange G), the reaction mixtures were resolved in 0.8% agarose gel in 1x TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA). The gels were scanned using Amersham Typhoon scanner (Cytiva) and quantified in ImageJ.