Aliquots from bacterial cultures with either arabinose or glucose were withdrawn and mixed with 0.25 volume of 95% ethanol, 5% phenol, and frozen in liquid nitrogen. After thawing on ice, cells were pelleted by centrifugation (15 min at 4000 g) and RNA extracted by hot-phenol (41 (link)). 10 μg of total RNA was mixed with 2 × RNA loading buffer (95% (v/v) formamide, 0.025% (w/v) bromophenol blue, 0.025% (w/v) xylene cyanol), denatured for 2 min at 90°C, and separated on a 6% sequencing gel. After electrophoresis, RNAs were transferred to an Amersham HybondTM-N+ membrane (GE Healthcare) by electro-blotting, and UV-crosslinked. 5′-end-labeled oligodeoxyribonucleotide probes were used for detection of tisB mRNAs (ced267) and the 5S rRNA (5S-long). Pre-hybridization and hybridization of the membrane was carried out in Church and Gilbert hybridization buffer (Church & Gilbert, 1984) at 50°C. Signals were detected using a PhosphorImager screen and a PMI scanner™ (Biorad).
Amersham hybond n membrane
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, China
Amersham Hybond-N+ membrane is a nylon-based material used for nucleic acid transfer and immobilization in various molecular biology techniques. It provides efficient binding and retention of DNA, RNA, and oligonucleotides during blotting procedures.
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Lab products found in correlation
Hybond, by Cytiva (224 mentions)
Bio dot apparatus, by Bio-Rad (26 mentions)
Trizol reagent, by Thermo Fisher Scientific (13 mentions)
Anti m6a antibody, by Synaptic Systems (12 mentions)
Methylene blue, by Sangon (10 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (8 mentions)
Amersham ecl prime, by Cytiva (8 mentions)
Trizol, by Thermo Fisher Scientific (7 mentions)
M6a antibody, by Synaptic Systems (7 mentions)
Ssc buffer, by Merck Group (7 mentions)
Cdp star, by Roche (7 mentions)
Anti m6a antibody, by Abcam (6 mentions)
Dynabeads mrna purification kit, by Thermo Fisher Scientific (6 mentions)
Whatman, by Cytiva (6 mentions)
Amersham imager, by Cytiva (5 mentions)
Ultrahyb oligo buffer, by Thermo Fisher Scientific (5 mentions)
Specific m6a antibody, by Merck Group (5 mentions)
Dig dna labeling kit, by Roche (4 mentions)
Amersham imager 600, by GE Healthcare (4 mentions)
Thermo ecl supersignal western blotting detection reagent, by Thermo Fisher Scientific (4 mentions)
233 protocols using amersham hybond n membrane
1
RNA Extraction and Northern Blotting
2
Mitochondrial RNA Detection by Northern Blot
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Total RNA from cultured cells was isolated using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. RNA (2 µg) was separated on a denaturing formaldehyde/formamide 1.2% agarose gel and transferred and UV-crosslinked onto Amersham HybondTM-N membrane (GE Healthcare). RNA was visualized using [32P]-radiolabeled probes targeting mitochondrial RNAs as indicated (see Supplementary Table 1 ).
3
Southern Blot Analysis of Bacterial Mutants
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Southern blots were performed using genomic DNA of the wild type and mutants digested with BamHI (∆wzy and ∆kpsM∆wzy), EcoRI (∆kpsM and ∆kpsM∆wzy), AvaII (∆wzx), NcoI (∆wzc, wzcTrunc and ∆wzc∆wzb), and/or MfeI (∆wzb and ∆wzc∆wzb) (Thermo Scientific). The DNA fragments were separated by electrophoresis on a 1% agarose gel and blotted onto Amersham HybondTM‐N membrane (GE Healthcare). Probes were amplified by PCR and labeled using the primers indicated in Supporting Information Table S2 and DIG DNA labeling kit (Roche Diagnostics GmbH) according to the manufacturer's instructions. Hybridization was done overnight at 60°C (∆wzc, and ∆wzc∆wzb) or 65ºC (∆wzy, ∆wzx, ∆kpsM, ∆kpsM∆wzy, ∆wzb, ∆wzc∆wzb, and wzcTrunc), and digoxigenin‐labeled probes were detected by chemiluminescence using CPD‐star (Roche Diagnostics GmbH) in a Chemi DocTM XRS+Imager (Bio‐Rad).
4
MSTN Protein Expression Analysis
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Total protein was extracted using the IP lysis buffer (Beyotime, Beijing, China), and the protein was quantified using an Infinite 200 PRO multimode reader (Tecan, Männedorf, Switzerland). An aliquot containing 50 µg of total protein was subjected to SDS-PAGE on a 12% acrylamide gel, and the proteins bands were electrophoretically transferred to an Amersham Hybond TM-N+ membrane (GE Healthcare, Waukesha, WI, USA). The N-terminal domain of MSTN, β-actin, and tubulin proteins were detected using a 1∶2000 dilution of an anti-MSTN (LifeSpan Biosciences, Seattle, WA, USA), anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), or anti-tubulin (Abcam, Cambridge, MA, USA) primary antibody, and primary antibody reactivity was detected using a 1∶5000 dilution of a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). The N-terminal domain of MSTN, β-actin, and tubulin protein bands were visualized using an enhanced chemiluminescence method (Thermo Scientific).
5
Northern Blot Analysis of miR1432
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Approximately 30 μg of total RNA was separated on 18% polyacrylamide denaturing gels, using rice miR1432 RNA oligonucleotide as marker. RNAs were transferred to Amersham HybondTM‐N+ membrane (GE Healthcare, Amersham, UK) and hybridized with a locked nucleic acid DNA oligonucleotide complementary to the miR1432 sequence, which had been labelled with γ‐32pATP. Blots were prehybridized and hybridized at 42 °C in 125 mm NaHPO4 (pH 7.2), 250 mm NaCl2, 7% SDS and 50% formamide, and washed at 42 °C twice with 2 × SSC, 0.2% SDS followed by a higher stringency wash of 1 × SSC, 0.1% SDS at 37 °C. Blots were imaged using an FLA‐5000 phosphorimager (Fuji Medical Systems Inc., Stamford, CT). U6 was used as a loading control. Sequence of probe is listed in Table S6 .
6
RNA Separation and Northern Blot Analysis
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RNA separation and Northern blot analysis were performed as previously described.50 (link) In short, 7 μg RNA was separated in 10 % polyacrylamide-urea-gels at 300 V and blotted to Amersham HybondTM-N+ membrane (GE Healthcare) for 2 h at 100 mA. Northern blot hybridizations were carried out with labeled oligonucleotides listed in Table S5.
7
Transcription Factor Binding Analysis
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The CYP6B6 HE1 element was prepared as the DNA probe for the electrophoretic mobility shift assay (EMSA). The negative control contained only HE1 probe without HaADH5 protein. For specific competition, excess unlabeled HE1 DNA was added. For non‐specific competition, a 300 bp non‐correlated sequence was added and denoted as bHLH that was a basic helix‐loop‐helix gene from Chenopodium glaucum. The EMSA/Gel‐shift kit (Beyotime) was used for the reaction. Protein‐bound probes were separated from free probes on 6% (w/v) non‐denaturing PAGE in Tris‐borate ethylenediaminetetraacetic acid buffer. All DNA bands were transferred onto Amersham Hybond‐N+ Membrane (GE Healthcare, Waukesha, WI, USA) using the electrode diverting method. DNA probe labeling and subsequent color detection were performed using the DIG High Prime DNA Labeling and Detection Starter Kit ΙΙ (Roche, Basel, Switzerland) according to the manufacturer's protocol. Finally, the membrane was detected using an ImageQuant LAS4000 imager.
8
Quantifying mRNA m5C Modifications
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The total RNA of cells was extracted with RNAiso Plus (9109, Takara), followed by using the Magnetic mRNA Isolation Kit (s1550s, New England Biolabs) to purify polyadenylated mRNA twice according to the manufacturer. After 5-min denaturation at 95°C, the same amounts of serially diluted mRNA were loaded onto an Amersham Hybond N + membrane (RPN303B, GE Healthcare). After UV crosslinking twice for 2 min each time, membrane was stained with Methylene blue (A610622-0025, Sangon Biotech) according to the protocols of the manufacturer and was washed by 1× PBST. After the scanning to indicate the loading RNA, membrane was blocked with 5% non-fat milk in 1×PBST for 1 h at room temperature and then was incubated with anti‐m5C antibody (A19841, ABclonal) overnight (4°C). Membrane was washed by 1×PBST and was incubated by HRP-conjugated Affinipure goat anti-rabbit IgG (A0277, Beyotime) diluted 1:5,000 for 1 h at room temperature.
9
Quantification of 5hmC and 5mC levels
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Genomic DNA was extracted from cells using TIANamp Genomic DNA Kit (Tiangen Biotech, DP304) and sonicated with Bioruptor Plus (Diagenode) for 15 cycles to obtain ~300 bp fragments. DNA was denatured with 0.1 M NaOH, incubated at 99 °C for 5 min and neutralized with 0.1 volume of 2M ammonium sulfate pH7.0. 5 μg DNA was then spotted onto Amersham Hybond-N+ membrane (GE Healthcare), air-dried and UV-crosslinked. The membrane was blocked in 10% milk in TBS containing 0.1% Tween 20 (TBST) overnight at 4 °C followed by primary 5hmC or 5mC antibody (diluted in 5% BSA in TBST) incubation for 1 hour at room temperature. After washing with TBST, membrane was incubated with HRP-conjugated secondary antibody for 1 h at room temperature and proceeded with ECL exposure and imaging. Dot intensity was quantified by ImageJ software.
10
Genomic DNA Extraction and Transgene Detection in Plants
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Genomic DNA was extracted from young leaves of greenhouse-grown plantlets using cetyltrimethylammonium bromide method17 . The presence of each of the transferred genes was confirmed by PCR with gene-specific primers (Supplementary Table 2 ).
For Southern blot, 30 μg of plant genomic DNA was digested overnight at 37°C by 100U of EcoRV, a restriction enzyme that cuts T-DNA constructs used in this study at a single position inside nnHispS coding region. After gel electrophoresis, digestion products were transferred onto Amersham Hybond-N+ membrane (GE Healthcare, UK) and immobilized. The DNA probe was constructed by PCR using cloned synthetic nnluz gene as the template and nnluz-specific primers listed inSupplementary Table 2 . Probe DNA was labeled with alkaline phosphatase using the AlkPhos Direct Labeling Kit (GE Healthcare, UK). Prehybridization, hybridization (overnight at 60°C) with alkaline phosphatase-labeled probe, and subsequent washings of the membrane were carried out according to the AlkPhos Direct Labeling Kit protocol. Detection was performed using Amersham CDP-Star detection reagent following the manufacturer’s protocol (GE Healthcare, UK). The signal from the membrane was accumulated on X-ray film (XBE blue sensitive, Retina, USA) in film cassette at room temperature for 24 hours. X-ray films were scanned on Amersham imager 600 (GE Healthcare Life Sciences, Japan).
For Southern blot, 30 μg of plant genomic DNA was digested overnight at 37°C by 100U of EcoRV, a restriction enzyme that cuts T-DNA constructs used in this study at a single position inside nnHispS coding region. After gel electrophoresis, digestion products were transferred onto Amersham Hybond-N+ membrane (GE Healthcare, UK) and immobilized. The DNA probe was constructed by PCR using cloned synthetic nnluz gene as the template and nnluz-specific primers listed in
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