First, biotin label was linked to the 3′ end of artificially synthesized single-stranded oligonucleotide probe containing binding site by DNA 3′ end biotin label kit (Beyotime, Shanghai, China). Second, double-stranded DNA probe with biotin label was obtained by annealing with artificially synthesized complimentary chain. Third, purified CkREV protein was incubated with probe with biotin label at a certain proportion while unlabeled double-stranded probe was used as cold probe. Fourth, native-PAGE was employed to separate samples before being transferred onto nylon membrane (Solarbio, Beijing, China) with positive charge through wet transformation method. Fifth, the nylon membrane was placed under ultraviolet crosslinker purple (UVP, Upland, USA) at 254 nm, 120 mJ/cm2 for 60 s. Last, colour development was employed on a completely cross-linked nylon membrane by EMSA chemiluminescence kit (Beyotime, Shanghai, China) for observation under chemiluminescence imager. Detailed steps complied with instructions of EMSA chemiluminescence kit (Beyotime, Shanghai, China).
Nylon membrane
Manufactured by Solarbio
Sourced in China
The Nylon membrane is a laboratory equipment used for filtration and separation processes. It is a thin, flexible material made of nylon that is designed to allow the passage of certain molecules or particles while retaining others, based on their size or other properties.
Automatically generated - may contain errors
Lab products found in correlation
Nylon membrane, by Analytik Jena (2 mentions)
Dna 3 end biotin label kit, by Beyotime (2 mentions)
Ultraviolet crosslinker purple, by Analytik Jena (2 mentions)
Emsa chemiluminescence kit, by Beyotime (2 mentions)
Nylon membrane, by Beyotime (2 mentions)
Trizol based method, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Dig northern starter kit, by Roche (1 mentions)
Dig high prime dna labeling and detection starter kit 1, by Roche (1 mentions)
Plant genomic dna kit, by CWBIO (1 mentions)
Hindiii, by Takara Bio (1 mentions)
Miniprep kit, by Merck Group (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nylon membrane, by GE Healthcare (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Methylene blue, by Solarbio (1 mentions)
5 protocols using nylon membrane
1
Biotin-Labeled Oligonucleotide Probe for Protein Binding
2
CircRNA Northern Blot Analysis Protocol
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CircRNA northern blot assay was basically performed according to the standard method provided by DIG Northern Starter Kit (Roche, Cat. No. 12039672910). 1 μg of total RNA was extracted from 293T cells through Trizol-based method (Life Technologies) and separated by 2% gels with formaldehyde. Gels with total RNA were blotted to a nylon membrane (Solarbio) by capillary transfer overnight. Total RNA on nylon membrane was fixed by baking at 120 °C for 0.5 h. After prehybridizing membrane with DIG Easy Hyb at 68 °C for 30 min, the membranes were hybridized with DIG-labelled probes at 68 °C overnight. The membranes were washed with 2✕ SSC, 0.1✕ SSC, wash buffer, blocking buffer and antibody buffer. Then membranes were exposed to imaging device for 5-20 mins. Data was analyzed using Image Lab software (Bio-Rad).
3
Verification of Hygromycin Resistance in G. tritici Transformants
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The wild strain and four transformants of G. tritici were grown on petri dishes containing PDA at 25 °C for seven days before they were harvested in sterile distilled water. The mycelia were then transferred to a triangular flask containing 100 mL PDB and incubated on a rotary shaker (175 rpm) at 25 °C for five days. The mycelia were collected by vacuum filtration, and genomic DNA was extracted by Plant Genomic DNA Kit (CWBIO, Beijing, China). The primers YzbF (5′-GATCGTTATGTTTATCGGCACT-3′) and YzbR (5′-TGGCGACCTCGTATTGG-3′) were used for testing and verifying PCR amplification of the hph gene fragment.
Southern blot analyses of transformants were performed with a DIG-High Prime DNA Labeling and Detection Starter Kit I (Roche, Shanghai, China) [38 (link)]. For Southern blotting of transformants, 15 μg DNA from the wild type and four transformants were digested overnight with HindIII (Takara, Dalian, China), and the digested DNA was electrophoresed on 0.8% agarose gel. DNA fragments were transferred onto a nylon membrane (Solarbio, Beijing, China) and hybridized with a labelled probe (514 bp) obtained by PCR amplification with primers Yzb-F/R from plasmid pSilent-1.
Southern blot analyses of transformants were performed with a DIG-High Prime DNA Labeling and Detection Starter Kit I (Roche, Shanghai, China) [38 (link)]. For Southern blotting of transformants, 15 μg DNA from the wild type and four transformants were digested overnight with HindIII (Takara, Dalian, China), and the digested DNA was electrophoresed on 0.8% agarose gel. DNA fragments were transferred onto a nylon membrane (Solarbio, Beijing, China) and hybridized with a labelled probe (514 bp) obtained by PCR amplification with primers Yzb-F/R from plasmid pSilent-1.
4
Quantification of m6A-Methylated mRNA
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Poly(A)+ mRNA was enriched using GenElute messenger RNA (mRNA) Miniprep Kit (Sigma‐Aldrich). The concentration and purity of RNA were measured by an Agilent Bioanalyzer 2100. Poly(A)+ mRNA samples were denatured at 70 °C for 5 min and then diluted equally to 400 ng, 200 ng and 100 ng in equal volumes. After denaturation, equal volume of diluted mRNA was added into a nylon membrane (GE Healthcare). The membranes were cross-linked at 245 nm UV for three times under auto-cross-linking mode (UVP analytik jena, CL-1000M). Then, the membranes were blocked with 5% BSA for 2 h at room temperature and incubated with anti‐m6A antibody (Synaptic System, 202003) overnight at 4 °C. Membranes were washed with PBST for three times. Horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (CST, 7074P2) were diluted 1:5000 and incubated with the membranes for 1 h at room temperature. Membranes were washed with PBST and the signals were detected by standard analysis of HRPO-induced chemiluminescence (ECL, Millipore). The same gradient-diluted Poly(A)+ mRNA was also added into the nylon membrane, stained with methylene blue (Solarbio, G1300) for 2 h, and washed with ribonuclease-free water.
5
Biotin-Labeled Oligo EMSA Protocol
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First, biotin label was linked to the 3' end of arti cially synthesized single-stranded oligonucleotide probe containing binding site by DNA 3' end biotin label kit (Beyotime, China). Second, double-stranded DNA probe with biotin label was obtained by annealing with arti cially synthesized complimentary chain. Third, puri ed CkREV protein was incubated with probe with biotin label at a certain proportion while unlabeled double-stranded probe was used as cold probe. Fourth, native-PAGE was employed to separate samples before transferred onto nylon membrane (Solarbio, China) with positive charge through wet transformation method. Fifth, the nylon membrane was placed under ultraviolet cross linker purple (UVP, USA) at 254 nm, 120 mJ/cm 2 for 60 s. Last, colour development was employed on completely crosslinked nylon membrane by EMSA chemiluminescence kit (Beyotime, China) for observation under chemiluminescence imager. Detailed steps complied with instructions of EMSA chemiluminescence kit from Beyotime Company.
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