Aliquots of 200–400 ng extra-chromosomal DNA were separated (50 volts for 60 min) on a 0.7 % agarose, 1X TAE gel. Invitrogen (Eugene, OR USA) SYBR Safe DNA gel stain (# S33102) was added to the gel at a 1∶10,000 dilution. DNA was treated and transferred from gel to Positively Charged Nylon Transfer Membrane (#RPN303B, GE Healthcare, UK) using standard Southern blot procedures [32] . Hybridization probes were prepared from PCR products generated using primers 5′-CCTGTTGCTTGGGTAACTGTATC-3′ and 5′-AATGGCAGAAAGTGGCTGG-3′ for identified bacteriophage in the NRS19 strain and primers 5′TGCCATTGTGATGAGGAGGG-3′ and 5′-GCAACGCAGATTGTTTGAGTG-3′ for the identified bacteriophage of the NRS26 strain. These PCR products were purified with QIAquick PCR Purification Kit # 28106 and labeled for use as a Southern blot probe using Biotin DecaLabel DNA Labeling Kit (#K0652, Thermo Scientific (Waltman, MA USA). DNA transferred to nylon membrane was pre-hybridized and hybridized based on standard Southern blot procedures [32] . Southern blot probe detection was performed using Fermentas (Glen Burnie, MD USA) Biotin Chromogenic Detection Kit (#K0661) based on manufacturer's instructions.
Positively charged nylon transfer membrane
Manufactured by GE Healthcare
Sourced in United Kingdom
The positively charged nylon transfer membrane is a lab equipment product designed for the transfer and immobilization of biomolecules, such as nucleic acids and proteins, onto a solid support. The membrane's positive charge facilitates the efficient binding of negatively charged biomolecules, enabling their subsequent detection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Scintillation counter, by Beckman Coulter (2 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit 28106, by Thermo Fisher Scientific (1 mentions)
Biotin decalabel dna labeling kit, by Thermo Fisher Scientific (1 mentions)
Biotin chromogenic detection kit, by Thermo Fisher Scientific (1 mentions)
Flagipt1, by Merck Group (1 mentions)
Chemiluminescent nucleic acid detection module, by Thermo Fisher Scientific (1 mentions)
Phosphorimager, by Bio-Rad (1 mentions)
Ultrahyb buffer, by Thermo Fisher Scientific (1 mentions)
Illustra microspin g 25 column, by GE Healthcare (1 mentions)
Rnase a, by Roche (1 mentions)
Anti nrf2 antibody, by Abcam (1 mentions)
Chemiluminescent emsa kit, by Beyotime (1 mentions)
Beyoecl star, by Beyotime (1 mentions)
Chromosomal grade, by Bio-Rad (1 mentions)
Chef mapper, by Bio-Rad (1 mentions)
Dig high prime dna labeling and detection starter kit 2, by Roche (1 mentions)
7 protocols using positively charged nylon transfer membrane
1
Bacteriophage DNA Detection via Southern Blot
2
RNA-Protein Binding Assay with m6A-RNA
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RNA electrophoretic mobility shift assay (EMSA) was performed by LightShift RNA EMSA optimization and control kit (Thermo Fisher) following the manufacturer’s instructions and a previous report46 (link). Briefly, IGF2BP1-Flag proteins, WT and G19A-mutated RBRP oncopeptides, were overexpressed and purified from HEK293T cells according to the kit instructions (FLAGIPT1, Sigma) as previously described41 (link). Biotin-labeled m6A-RNA oligos (20 pmol), 6 μg recombined IGF2BP-Flag proteins, together with 2 μg recombined RBRP-Flag oncopeptides or RBRP-Flag G19A mutant oncopeptides were mixed and incubated at room temperature for 30 min. Then, 1 µl of 0.2% glutaraldehyde was added to the mixtures and incubated on ice for 15 min. Then, the RNA-protein/oncopeptide mixtures were separated on 6% TBE (450 mM Tris, 450 mM boric acid, 10 mM EDTA pH 8.3) gel. The gel was transferred to a positively charged nylon transfer membrane (GE Healthcare) and the RNA membrane was crosslinked by UV light. The biotin-labeled m6A-RNA oligos were detected by a chemiluminescent nucleic acid detection module (Thermo Fisher) following the manufacturer’s instructions. These A- or m6A-oligo sequences were provided in Supplementary Table 3 .
3
Quantifying Telomeric and snRNA Levels
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10 μg of RNA resuspended in SSC and formaldehyde were denatured at 70°C and then dot-blotted on a positively charged nylon transfer membrane (GE Healthcare), and UV crosslinked with a UV-stratalinker (Stratagene). TERRA and U2 were detected using a (CCCTAA)5 or a U2 specific oligonucleotide probe labeled with 32P-γ-ATP by T4 polynucleotide kinase (NEB) and purified with illustra microspin G-25 columns (GE Healthcare). Hybridizations were performed using UltraHyb buffer (Ambion) for 16–18 h at 43°C or 50°C. Membranes were washed in 2× SSC/0.1% SDS for 10 min at room temperature and in 0.2× SSC/0.1% SDS for 20 min at 50°C. Blots were stripped for 10 min with 0.1× SSC, 40 mM Tris (pH 7.5), and 1% SDS buffer preheated at 80°C. When indicated, RNA samples were treated with RNaseA (Roche) at a final concentration of 100 μg/ml for 30–60 min at 37°C. Images were captured with a Phosphorimager (BioRad) and signals were quantified using ‘Quantity one’ software.
4
Electrophoretic Mobility Shift Assay for Nrf2 Binding
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Synthesized forward and reverse strand oligonucleotides of the putative ARE in GPX7 promoter region were hybridized to form double‐stranded DNA probes. The 5′ of forward strand was labeled by biotin. Wild‐type and mutant HO1‐ARE without biotin label were used as the competitor. Binding reactions were carried out using Chemiluminescent EMSA Kit (Beyotime) according to the manufacturer's instructions. For supershift analysis, extracts were pre‐incubated for 20 min on ice with anti‐Nrf2 antibody (Abcam, ab62352). DNA‐binding protein complexes were separated by nondenaturing 5% PAGE in 0.5× Tris‐Borate‐EDTA buffer and subsequently electrotransferred onto a positively charged nylon transfer membrane (GE Healthcare). Blots were developed by horseradish peroxidase‐labeled streptavidin and BeyoECL Star (Beyotime). All the sequences designed for EMSA are listed in Appendix Table S5 .
5
Microtubule-dependent kinetics of TTLL6 and TTLL4
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The KM and kcat values for TTLL6 wild-type, the switch mutant (C179A/Q180R/H362I) and TTLL4 with brain and unmodified microtubules were determined by varying the concentration of tubulin from 1 to 20 µM for TTLL6 and 1 to 30 µM for TTLL4 while keeping the enzyme concentration constant at 1 µM. The reaction condition consisted of 50 mM Hepes pH 7.0, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 1 mM ATP, 950 µM L-Glutamic acid, and 50 µM H 3 -L-Glutamic acid. Reactions were initiated by the addition of the enzyme and terminated by the addition of saturating amounts of EDTA and placing it on ice for 30 mins. The reaction mix was then transferred to the positively charged nylon transfer membrane (GE Lifesciences) and washed extensively with 50 mM sodium phosphate, pH 7.8 supplemented with 25 mM KCl. The membranes were then transferred to a scintillation vial and residual radioactivity was measured using a Beckmann scintillation counter. Kinetic constants were obtained by using the one site binding parameter equation in Prism 6.1. Reactions were performed in duplicate on two separate days.
6
Pulsed-field Gel Electrophoresis of Yeast Chromosomes
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Chromosomal DNA was prepared in agarose plugs as described previously27 (link). Plugs were washed in TE and inserted into wells of 1.1% agarose (Chromosomal grade, Bio-Rad) gel prepared in 0.5 × TBE buffer. The chromosomes were separated by pulse-field electrophoresis in 0.5 × TBE with a CHEF-Mapper apparatus (Bio-Rad). The running parameters were: 200 V, linear ramping from 40 to 120 s for 26 h at 14 °C. The chromosomal bands were visualized by staining with ethidium-bromide and destaining in sterile water. DNA blotting on positively charged nylon transfer membrane (GE Healthcare) was performed as described before24 (link). Y’ sequence PCR product was labelled with DIG High Prime DNA Labeling and Detection Starter KitII (Roche). The labelled DNA was hybridised to the membrane overnight at 68 °C after 30 min prehybridisation. After hybridisation the membrane was washed first at room temperature in 2 × SSC, 0.1% SDS and then twice at 68 °C in 0.1 × SSC, 0.1% SDS.
7
Microtubule-dependent kinetics of TTLL6 and TTLL4
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The KM and kcat values for TTLL6 wild-type, the switch mutant (C179A/Q180R/H362I) and TTLL4 with brain and unmodified microtubules were determined by varying the concentration of tubulin from 1 to 20 µM for TTLL6 and 1 to 30 µM for TTLL4 while keeping the enzyme concentration constant at 1 µM. The reaction condition consisted of 50 mM Hepes pH 7.0, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 1 mM ATP, 950 µM L-Glutamic acid, and 50 µM H 3 -L-Glutamic acid. Reactions were initiated by the addition of the enzyme and terminated by the addition of saturating amounts of EDTA and placing it on ice for 30 mins. The reaction mix was then transferred to the positively charged nylon transfer membrane (GE Lifesciences) and washed extensively with 50 mM sodium phosphate, pH 7.8 supplemented with 25 mM KCl. The membranes were then transferred to a scintillation vial and residual radioactivity was measured using a Beckmann scintillation counter. Kinetic constants were obtained by using the one site binding parameter equation in Prism 6.1. Reactions were performed in duplicate on two separate days.
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