Total cellular RNAs were extracted using Trizol reagent (Thermo) according to the manufacturer’s instructions. For the detection of EGFP mRNA, 5 μg of total RNAs were subjected to denatured 1.5% agarose gels with 2.2 mol/L formaldehyde. The separated RNAs were transferred onto the Hybond-A nylon membrane (GE Healthcare), and fixed at 120 °C for 15 min. Then the membranes were hybridized with DIG-labeled probes in Hybridization Ovens at 65 °C overnight. Finally, the membranes were incubated with anti-DIG antibody conjugated with alkaline phosphatase and exposed to the luminescent image analyzer LAS4000 (Fuji Film). The probes for detection of EGFP and GAPDH mRNA were complementary to their ORF region of 500–720 nt and 760–1060 nt, respectively. For detection of small RNAs, 20 μg of total RNAs were subjected to 7 mol/L urea-15% PAGE and transferred to Hybond-A nylon membrane (GE Healthcare). The membrane was chemically cross-linked in 1-ethly-3-(3-dimethylaminopropyl) carbodiimide (EDC) at 60 °C for 30 min. The DIG-labeled RNA probes targeting EGFP siRNA and U6 were synthesized by Takara.
Hybond a nylon membrane
Manufactured by GE Healthcare
Sourced in United Kingdom
Hybond-A is a nylon membrane used in laboratory applications. It is designed for the transfer and immobilization of nucleic acids such as DNA and RNA from electrophoresis gels for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Dig utp, by Roche (2 mentions)
Cdp star, by Roche (2 mentions)
Anti dig alkaline phosphatase antibody, by Roche (2 mentions)
X ray film, by Fujifilm (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Las 4000, by Fujifilm (1 mentions)
Dig rna labeling mix, by Roche (1 mentions)
Rnase inhibitor, by Promega (1 mentions)
4 protocols using hybond a nylon membrane
1
RNA Extraction and Northern Blot Analysis
2
Gel Shift Assay for RNA-Protein Interactions
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A gel mobility shift assay was performed in 50 mM HEPES–KOH (pH 8.0), 100 mM NaCl, 2 mM MgCl2, 1 mM tRNA, 2 mM DTT, and 20 U RNasin, at a total volume of 10 μL reaction with the indicated amount of proteins and 0.1 pmol double-strand RNA (dsRNA) or single-strand RNA (ssRNA). The dsRNA was labeled with DIG-UTP (Roche, Basel, Switzerland) by in vitro transcription and derived from 1 to 200 nt of EGFP. Reactions were incubated for 30 min at 25 °C. The reactions were terminated by the addition of 2.5 μL 5× sample buffer (20 mM Tris–HCl (pH 8.0), 30% glycerol and 0.1% bromophenol blue). The nucleic acid–protein complexes were separated by electrophoresis on 1.5% agarose gels and transferred to the Hybond-A nylon membrane (GE Healthcare, Little Chalfont, Buckinghamshire, UK). After that, the membrane was subjected to cross-linking at 120 °C and was incubated with anti-DIG-alkaline phosphatase antibody (Roche, Basel, Switzerland), followed by incubating with CDP-STAR (Roche, Basel, Switzerland) for 15 min at 37 °C. The signals were then detected by X-ray film (Fujifilm, Tokyo, Japan).
3
Gel Shift Assay for RNA-Protein Interactions
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Gel mobility shift assay was performed in 50 mM HEPES–KOH (pH 8.0), 100 mM NaCl, 2 mM MgCl2, 1 mM tRNA, 2 mM DTT, 20 U RNasin, in a total volume of 10 μl reaction with the indicated amount of proteins and 0.1 pmol dsRNA or ssRNA. The dsRNA and ssRNA were labeled with DIG-UTP (Roche) by in vitro transcription and derived from 200-nt EGFP. Reactions were incubated for 30 min at 25°C. The reactions were terminated by the addition of 2.5 μl 5× sample buffer [20 mM Tris–HCl (pH 8.0), 30% glycerol and 0.1% bromophenol blue]. The nucleic acid–protein complexes were separated by electrophoresis on 1.5% agarose gels and transferred to Hybond-A nylon membrane (GE Healthcare). After that, the membrane was subjected to cross-linking with 120°C and was incubated with anti-DIG-alkaline phosphatase antibody (Roche), followed by incubating with CDP-STAR (Roche) for 15 min at 37°C. The signals are then detected by X-ray film (Fujifilm, Tokyo, Japan).
4
RNA-Protein Binding Assay
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We generated 200-nt DIG-labeled dsRNA and 22-nt siRNA via in vitro transcription using DIG RNA labeling mix (Roche). MBP-fusion NS2 or mutant proteins were reacted with DIG-labeled RNAs (0.2 μmol/L 200-nt dsRNA or 22-nt siRNA) in a binding buffer [50 mmol/L HEPES (pH 8.0), 15 mmol/L NaCl, 0.5 mmol/L MgCl2, 10% glycerol and 1 U of RNase inhibitor (Promega)] at 22 °C for 45 min; the total volume was 10 μL. Then the reaction mixtures were separated on 6% (for dsRNA) or 12% (for siRNA) TBE-PAGE and transferred to Hybond-A nylon membrane (GE Healthcare). The membranes were incubated for 30 min with anti-DIG antibody conjugated with alkaline phosphatase (Roche).
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