DNA fragments harboring eryAI, ermE, or SACE_3447 promoter regions were independently obtained by PCR amplification with their respective primers (Table S1 ), and 5′-end labeled with 6-carboxyfluorescein (6-FAM). Those labeled DNA fragments were successively mixed with above purified His6-tagged SACE_3446 proteins. The binding reaction system contained 10 mM Tris (pH 7.5), 5 mM MgCl2, 50 mM EDTA, 60 mM KCl, 10 mM DTT, 10% glycerol, 150 ng labeled probe, and 0.2–1.4 µM purified protein. 7.5 µg unlabeled DNA fragments or 7.5 µg poly dIdC were respectively added into the reaction system for competitive assays. After incubation on ice for 10 min, the reactants were run on an 8% TBE polyacrylamide gel (Bio-Rad) buffered in 0.5 X TBE at 30 mA for 1 h.
Tbe polyacrylamide gel
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The TBE polyacrylamide gel is a laboratory equipment used for the electrophoretic separation of biomolecules, such as DNA, RNA, or proteins. It provides a high-resolution medium for the analysis and purification of these macromolecules.
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Lab products found in correlation
Sybr safe dna gel stain, by Bio-Rad (1 mentions)
Cas9prot 250ug, by Merck Group (1 mentions)
Bluejuice loading buffer, by Thermo Fisher Scientific (1 mentions)
Geldoc xrs, by Bio-Rad (1 mentions)
Sybr gold, by Thermo Fisher Scientific (1 mentions)
Chemiluminescent emsa kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using tbe polyacrylamide gel
1
SACE_3446 Protein Binding Assay
2
gDNA Extraction, Amplification, and Digestion
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gDNA extraction, target amplification, and purification were performed identically to the T7E1 assay. Digestion with corresponding restriction enzymes was fulfilled according to the manufacturer’s instructions using 300 ng purified product and was subsequently resolved on a 4%–20% high-resolution Tris-borate-EDTA (TBE) polyacrylamide gel (no. 4561093S; Bio-Rad, Hercules, CA, USA), following staining with SYBR Safe DNA Gel Stain.
3
EMSA analysis of eryAI and ermE promoters
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EMSAs were carried out in the light of previously described methods [47 (link)]. Using the genomic DNA from S. erythraea A226 as a template, the promoter regions of eryAI and ermE were successively amplified by PCR with their respective primers (Table 2 ) labeled at 5′ end by the 6-isomer of carboxyfluorescein (6-FAM). Those labeled DNA fragments were independently mixed with purified His6-tagged SACE_7301 or BldD proteins. The binding reaction system consisted of 10 mM Tris (pH 7.5), 5 mM MgCl2, 50 mM EDTA, 60 mM KCl, 10 mM DTT, 10% glycerol, 150 ng labeled probes and 0.5-4 μM purified SACE_7301 or BldD. For competitive inhibition of the binding reaction, 7.5 μg unlabeled above-mentioned DNA fragments or 7.5 μg poly dIdC were added into that reaction system, respectively. After incubation on ice for 10 min, the reactants were run on an 8% TBE polyacrylamide gel (Bio-Rad) with 0.5 × TBE as a running buffer at 30 mA for 1 h.
4
Exploring EdeB Binding to ede Promoter
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EMSA was conducted to explore whether EdeB directly binds to the promoter region of ede BGC according to the previous method (Hellman and Fried, 2007 (link)). Briefly, the edeB gene was amplified from genomic DNA of wild-type B. brevis X23 using primers edeB-F and edeB-R. The vector pET28a was digested by Xba I and Xho I, and then incubated with purified amplicons of edeB. Then, the mixture was electroshocked into Escherichia coli GB05-dir containing the recombinant system through homologous recombination. The extracted plasmid was identified and transformed into competent cells BL21(DE3) to obtain recombinant strain BL21/pET28a-edeB. The EdeB protein was purified from the culture after IPTG induction. The promoter region edePro of ede BGC was amplified by PCR using primer pairs edePro-F/edePro-R with 6-FAM at the 5’end (Supplementary Table 1
5
CRISPR RNP Gel Shift Assay
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10 pmol of purified sgRNA (IDT, Alt-R CRISPR-Cas9 sgRNA) alone, or 10 pmol of sgRNA and 10 pmol of purified spCas9 nuclease (Sigma-Aldrich, CAS9PROT-250UG), were incubated at room temperature for 10 min in a 20 μl reaction in 1 × 3.1 Buffer (B7203S, NEB) to form RNP. Specified dose of TAMRA dye labelled PNA was diluted into 1 μl of water, added to wells, and incubated in a thermocycler for 30 min at 37°C. A BIORAD 5% TBE polyacrylamide gel was pre-run for 30 min at 10 mA. 5 μl of final binding reactions were loaded onto gels with BlueJuice loading buffer (Invitrogen) and run at 10 mA. Gels were first imaged using BIORAD GelDoc XRS+ with a Green Epi (BIORAD #170-8284) 605/50 filter to acquire TAMRA signal and then incubated in 1× SYBR Gold (Invitrogen) in 1× TBE buffer for 5 min before imaging by standard UV transillumination (Raw images in Supplementary Figure S1 ). For band density analysis, three independent experiments were loaded onto a single 26-well gel and run according to the specifications above. Integrated densities for TAMRA-Cas9 co-localized bands were determined across wells using ImageJ analyses, and background signal from each no PNA (0 pmol) control was subtracted for each experiment.
6
Electrophoretic Mobility Shift Assay for Nrf2
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EMSA was performed using the commercial Chemiluminescent EMSA kit (Pierce Biotechnology). For EMSA, 5 mg of total extracted nuclear proteins was incubated with 1 pmol double stranded ATP end-labeled oligonucleotide probe containing the sequences in binding buffer (10 mM HEPES, pH 7.9, 80 mM NaCl, 3 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10% glycerol). After the incubation, the samples were loaded on a 5% TBE-polyacrylamide gel (Bio-Rad) and electrophoretically separated in 0.5× TBE buffer. Levels of Nrf2 DNA binding activity were quantified by computer-assisted densitometric analysis.
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