Reaction mixtures contained (7 μl): 10 μM E. coli RNAP holoenzyme, 7 μM PcopA derivative promoter DNA, 0 or 28 μM CueR in 10 mM Hepes pH 7.5, 50 mM KCl, 5 mM MgCl2, 3 mM DTT, and 100 μM AgNO3. Reaction mixtures were incubated for 1h on ice and followed by heparin challenge (100 μg/ml; final concentration) when indicated. The complexes were separated by 5% TBE gel in the TBE buffer (90 mM Tris-borate, pH 8.0, and 2 mM EDTA), stained using SYBR-Gold, and analyzed by Tanon-2500 (Tanon Science & Technology Co., Ltd).
Tanon 2500
Manufactured by Shanghai Tanon Science & Technology
Sourced in China
Tanon-2500 is a protein electrophoresis system designed for high-resolution separation of proteins. It features a large-format vertical gel system and can accommodate gels up to 20 x 20 cm in size. The system is suitable for a variety of protein electrophoresis techniques, including SDS-PAGE, native PAGE, and isoelectric focusing.
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6 protocols using tanon 2500
1
Transcriptional Activation of CueR Regulator
2
Gel-shift Assay of Bacterial RNAP
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Nucleic-acid scaffolds (sequences are listed in Supplementary Fig. 1a ) for the gel-shift assay were prepared as above. Reaction mixtures contained (4 μl): 0.75 μM B. subtilis RNAP holoenzyme, 0.5 μM Pbmr-2 or Pbmr-wt1 promoter DNA, 0 or 2 μM BmrR, 0 or 500 μM TPP in 10 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, and 3 mM DTT. Reaction mixtures were incubated for 1 h at room temperature and followed by heparin challenge (100 μg/ml; final concentration) when indicated. The complexes were separated by 5% TBE gel in the TBE buffer (90 mM Tris-borate, pH 8.0, and 2 mM EDTA), stained using SYBR-Gold, and analyzed by Tanon-2500 (Tanon Science & Technology Co., Ltd).
3
Stability Assessment of Cur@PHBX-PR/FUdR Nanospheres
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Stability assays of Cur@PHBX-PR/FUdR15S in BSA and different pH were conducted in various simulated in vivo conditions. Cur@PHBX-PR/FUdR15S was incubated in 10% unheated FBS for 1, 2, 4, 8, and 12 h and in acidic (pH 4.5), neutral (pH 7.4), and basic (pH 8.0) buffer solutions at 37 °C for 2.0 h. Subsequently, these PHBX nanospheres were also incubated in acidic buffer (pH 4.5) with 20 U/mL DNase II for 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 h. Samples were loaded onto 2% agarose gel for electrophoresis under 100 V in TAE buffer. The gel was stained using the GelStain and visualized using the Tanon 2500 (Tanon Science & Technology Co., Ltd., Shanghai, China).
4
Evaluating Affi-F/GQ Stability in Vitro
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The stability assays of affi-F/GQs were conducted in simulated various in vivo conditions. The affi-F/GQs were incubated in different pH buffers (acidic: pH = 4.5, neutral: pH = 7.4 and alkaline: pH = 9.0) at 37 °C for 2.0 h. Subsequently, DNA micelles were also treated with 0.36 U/mL DNase I for 1.0 h, 2.0 h, 4.0 h and 8.0 h. Finally, they were dissolved into different concentration of BSA (1, 10, 100 and 1000 µM) for 1.0 h. Then, the processed sample was loaded onto 2% agarose gel for electrophoresis, and the gel was stained by GelStain and photographed by Tanon 2500 (Tanon Science & Technology Co., Ltd., Shanghai, China).
5
Genotyping HO-1 Knockout Mice
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HO-1+/− and wild-type (HO-1+/+) mice (n=7 in each group) were provided by Jackson Laboratory. DNA from mouse tail tips were extracted using a Genomic DNA Extraction kit from Accurate Biotechnology Co., Ltd. (cat. no. AG21009). The DNA was used as a template to perform the PCR assay. PCR was performed with 10 µl Taq 2X PCR master mix (K0171, Thermo Fisher Scientific, Inc.), 2 µl mixed primers (10 µM of primer each), 1 µl genomic DNA and 7 µl ddH2O. Target DNA was amplified in a thermocycler machine (ABI Veriti), with initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 20 sec, annealing at 58°C for 30 sec, and extension at 72°C for 30 sec. The PCR products were electrophoresed on a 2% TBE Agarose gel and imaged in a Gel Imaging System (Tanon 2500, Tanon Science & Technology Co., Ltd.). Primers P1 (GTA CAT GCT GGC TGG GTT CT), P2 (CCA TTT CTC AGG CAA GAA GG) and P3 (GCC AGA GGC CAC TTG TGT AG) were used to identify HO-1+/+ [size, 280 base pair (bp)] and HO-1+/− (size, 280 and 225 bp) mice.
6
Quantification of miR-21a-5p in MSCs-EVs
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RNA was extracted from the MSCs-EVs and cDNA was synthesized in reverse transcription polymerase chain reaction (RT-PCR) as described above. The cDNA for miR-21a-5p was then amplified using PCR with specific primers (Table 3
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