Immobilon ny membrane

Manufactured by Merck Group

Sourced in United States, United Kingdom, Switzerland

Immobilon-NY+ membrane is a nylon membrane used for nucleic acid transfer and immobilization in various molecular biology applications. It is a positively charged membrane that can effectively bind nucleic acids, allowing for efficient transfer and detection of DNA and RNA samples.

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Lab products found in correlation

Amersham typhoon rgb biomolecular imager, by GE Healthcare (2 mentions) Benzonase, by Merck Group (2 mentions) Typhoon fla 9000, by GE Healthcare (2 mentions) Cdp star kit, by GE Healthcare (2 mentions) T4 polynucleotide kinase, by New England Biolabs (2 mentions) Dig dna labeling and detection kit, by Roche (2 mentions) Ultrahyb, by Thermo Fisher Scientific (2 mentions) Dig northern starter kit, by Roche (2 mentions) Dig wash and block buffer kit, by Roche (1 mentions) Anti digoxigenin ap, by Roche (1 mentions) Cdp star, by Merck Group (1 mentions) Imagequant las 4000, by GE Healthcare (1 mentions) Isogen, by Nippon Gene (1 mentions) Fuji fla 7000, by Fujifilm (1 mentions) Trans blot turbo apparatus, by Bio-Rad (1 mentions) Phosphorimager screen, by GE Healthcare (1 mentions) Detection starter kit 1, by Roche (1 mentions) Hipure bacterial dna extraction kit, by Magen Biotechnology Co (1 mentions) Chemiluminescent emsa kit, by Beyotime (1 mentions) Bacterial genomic dna miniprep kit, by Corning (1 mentions)

Close spelling variants of this product

Immobilon-Ny+ Transfer Membrane (3 citations) IMMOBILON NY+ charged nylon membrane (2 citations) Immobilon-Ny+ nylon membrane (5 citations) IMMOBILON-NY+, (20 citations) Immobilon membrane (188 citations) Immobilon (435 citations)

34 protocols using immobilon ny membrane

Mouse Liver Total RNA Expression Analysis

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Total RNA was extracted from mouse livers using ISOGEN (Nippon Gene). For analyzing mt-tRNA expression levels, total RNA was separated by electrophoresis through a 7 M urea 10% acrylamide gel. For aminoacyl-tRNA analysis, total RNA was extracted and electrophoresed on a 7 M urea 8% acrylamide gel under acidic conditions (pH 5.0) and then transferred to Immobilon-Ny + membranes (Merck). For expression level analysis of precursor RNAs, total RNA was separated by electrophoresis on a formaldehyde–2% agarose gel along with DynaMarker RNA High (BioDynamics Laboratory Inc.), and then transferred to Immobilon-Ny + membranes (Merck). The membranes were hybridized with DIG-modified probes that bind to mt-tRNA^Leu(UUR), mt-tRNA^Ile, 5.8S rRNA, ND1, 16S rRNA and 18S rRNA (the probe sequences are listed in Supplemental Table S5). The membranes were then blocked using a DIG wash and Block buffer kit (Roche) and incubated with anti-Digoxigenin-AP (1:10 000; #11093274910; Roche). Signals were detected using the ImageQuant LAS4000 (GE Healthcare) using CDP-star (Sigma-Aldrich).

Tani H., Ishikawa K., Tamashiro H., Ogasawara E., Yasukawa T., Matsuda S., Shimizu A., Kang D., Hayashi J.I., Wei F.Y, & Nakada K. (2022). Aberrant RNA processing contributes to the pathogenesis of mitochondrial diseases in trans-mitochondrial mouse model carrying mitochondrial tRNALeu(UUR) with a pathogenic A2748G mutation. Nucleic Acids Research, 50(16), 9382-9396.

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tRNA and RNA Separation and Detection

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0.4 μg of bulk tRNA or 1 μg of total RNA per sample were heated for 5 min at 80 °C and applied onto denaturing polyacrylamide gels (8 % polyacrylamide (19:1), 7 M urea, with or without 0.05 % ([N-acryloylamino]phenyl)mercuric chloride (APM) in 0.5 × TBE) (31 (link)). RNA was transferred onto Immobilon NY⁺ membranes (Millipore) by semi-dry transfer with 0.5× TBE. Northern blot was performed essentially as described using specific DNA-oligo probes (21 (link)). Signals were collected and analysed using a Fuji FLA 7000 phosphoimager (Fujifilm). Digital image analysis was performed using Fiji-ImageJ2 (The Fiji Project).

Alings F., Scharmann K., Eggers C., Böttcher B., Sokołowski M., Shvetsova E., Sharma P., Roth J., Rashiti L., Glatt S., Brunke S, & Leidel S.A. (2023). Ncs2* mediates in vivo virulence of pathogenic yeast through sulphur modification of cytoplasmic transfer RNA. Nucleic Acids Research, 51(15), 8133-8149.

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Quantitative DNA Slot Blot Assay for AAV Vector Titers

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The physical genomic titers of highly purified scAAV vector stocks were determined by quantitative DNA slot blot analysis as described previously [48 (link)], with modification. Briefly, 10 μl of vector stock was digested with Benzonase (EMD Millipore, Darmstadt, Germany) at 37°C for 1 hour. An equal volume of 100 mM NaOH was added, followed by incubation at 65°C for 30 min. A known quantity of plasmid DNA was denatured in the same manner for use as a reference standard for quantitation. Denatured DNA samples were loaded in two-fold serial dilutions onto Immobilon-NY+ membranes (Millipore, Bedford, MA). After UV cross-linking, the membranes were prehybridized for 1 hour at 42°C in a hybridization solution containing 6×SSC, 100 μg/ml denatured herring sperm DNA, 0.5% sodium dodecyl sulfate (SDS), and 5×Denhardt’s reagent. Subsequently, the membranes were hybridized with Cy3-labled DNA probe in a hybridization solution at 42°C for 18 to 20 hours. Membranes were washed twice with wash solution I (2×SSC, 0.1% SDS) at room temperature for 15 min, twice with wash solution II (0.5×SSC, 0.1% SDS) at 42°C for 15 min, and then exposed to Amersham Typhoon RGB Biomolecular Imager (GE Healthcare, Chicago, IL) at room temperature.

Yin L., Keeler G.D., Zhang Y., Hoffman B.E., Ling C., Qing K, & Srivastava A. (2020). AAV3-miRNA vectors for growth suppression of human hepatocellular carcinoma cells in vitro and human liver tumors in a murine xenograft model in vivo. Gene therapy, 28(7-8), 422-434.

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Northern Blot Analysis of tRNA Isoforms

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For each sample, 0.5 µg total RNA was separated on denaturing gels (10% polyacrylamide in 7 M urea and 1×TBE). The RNA was transferred to Immobilon NY+ membranes (Millipore) in 1×TBE at 4 mA cm⁻² for 40 min using a TransBlot Turbo apparatus (Bio-Rad) and crosslinked at 0.04 J in a Stratalinker ultraviolet light crosslinker. The membranes were incubated at 80 °C for 1 h and pre-hybridized at 55 °C for 4 h in hybridization buffer (20 mM Na₂HPO₄ pH 7.2, 5×SSC, 7% SDS, 2×Denhardt’s solution and 40 μg ml⁻¹ sheared salmon sperm DNA). This was followed by overnight hybridization with 10 pmol 5′-end ³²P-labelled probes (tRNA-Gly-CCC-2, 5′-CGGGTCGCAAGAATGGGAATCTTGCATGATAC-3′; tRNA-Arg-UCU-4, 5′-CGGAACCTCTGGATTAGAAGTCCAGCGCGCTCGTCC-3′ and tRNA-Asn-GUU-1, 5′-CGTCCCTGGGTGGGATCGAACC-3′) in hybridization buffer. Finally, the membranes were washed three times in 25 mM Na₂HPO₄ pH 7.5, 3×SSC, 5% SDS and 10×Denhardt’s solution, washed once in 1×SSC and 10% SDS, and exposed to PhosphorImager screens scanned on a Typhoon FLA 9000 (GE Healthcare). The band intensity was quantified using ImageJ.

Gao L., Behrens A., Rodschinka G., Forcelloni S., Wani S., Strasser K, & Nedialkova D.D. (2024). Selective gene expression maintains human tRNA anticodon pools during differentiation. Nature Cell Biology, 26(1), 100-112.

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Genetic Profiling of Xanthomonas oryzae

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Genomic DNA of 50 Xoo strains was extracted using the HiPure Bacterial DNA Extraction Kit (Magen, Guangzhou, China). DNA samples (50 µl) were digested with BamHI for 4 h at 37℃. The resulting DNA fragments were separated in 1.3% agarose gels at 80 V for 22 h and transferred to Immobilon-Ny + membranes (Millipore, USA). A hybridization probe was made from a digoxigenin (DIG)-labeled 2892-bp fragment derived from an SphI-digest containing the repetitive sequence of pthXo1 (GenBank: AY495676). The Detection Starter Kit I (Roche, Switzerland) was used to visualize hybridizing fragments according to the manufacturer’s instructions. The TALE-free strain Xoo PH containing an introduced copy of pthXo1, pthXo2 or avrXa7 (Additional file 1: Table S6) was used to locate the major tal genes involved in virulence.

Liu L., Li Y., Wang Q., Xu X., Yan J., Wang Y., Wang Y., Shah S.M., Peng Y., Zhu Z., Xu Z, & Chen G. (2024). Constructed Rice Tracers Identify the Major Virulent Transcription Activator-Like Effectors of the Bacterial Leaf Blight Pathogen. Rice, 17, 30.

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Confirm dsRNA Authenticity via Northern Hybridization

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Northern hybridization was performed to confirm the authenticity of the cDNA sequences generated from dsRNA-1 (6.2 kb) and dsRNA-2 (5.9 kb) in strains GZJS19 of L. biglobosa and t-459-V of B. cinerea. Two DNA probes, nt positions 1228–1697 for Probe 1 and nt positions 2318–2957 for Probe 2 (Table S3), were designed based on full-length cDNA sequences of dsRNA-1 and dsRNA-2, respectively. The purified dsRNA-1 and dsRNA-2 were separated in 1% (w/v) agarose gel and transferred to Immobilon-Ny⁺ membranes (Millipore, Bedford, MA, USA) by the capillary transfer method using 20 × SSC as transfer buffer [62 (link)]. Probe 1 and Probe 2 were pre-labeled as described by the manufacturers (GE Healthcare, Little Chalfont, United Kingdom) for hybridization with the denatured dsRNAs blotted on two membranes, respectively. The chemiluminescent signals of the probe-RNA hybrids were detected by using a CDP-Star kit (GE Healthcare Life Sciences, China).

Deng Y., Zhou K., Wu M., Zhang J., Yang L., Chen W, & Li G. (2022). Viral cross-class transmission results in disease of a phytopathogenic fungus. The ISME Journal, 16(12), 2763-2774.

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Measuring DNA-Polymerase Binding Kinetics

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Each 50 fmol of 5′-Biotin labeled oligonucleotide Bio-Olig2.5 (please see Table 1 for sequences of Bio-Olig2.5) was mixed with 1 U of Taq, Q5, GXL or 150 ng of KOD in 1X Binding Buffer (10 mM Tris-HCl pH 7.9, 50 mM KCl, 1 mM EDTA, 5% glycerol, 1 mM DTT, and 25 μg/ml BSA), and 50 pmol of N10 and MutL2-16 was used as specific competitor and non-specific competitor, respectively. The binding reaction was performed without MgCl₂ and Poly (dI-dC). After 20 min incubation at room temperature, the samples were mixed with 5X Loading Buffer (0.25X TBE buffer, 30% glycerol, and 0.2% bromophenol blue) and loaded onto 6% polyacrylamide gels in 0.5X TBE. To measure the equilibrium dissociation constant (KD) of DNAP and Olig2.5 interaction, 2 nM Bio-Olig2.5 was mixed with various concentration of purified KOD or Taq in a total volume of 20 μl^{25 (link)}. Oligonucleotides were electrophoresed and transferred onto Immobilon-Ny⁺ membranes (Millipore). Chemiluminescent detection was performed according to the user manual of Chemiluminescent EMSA Kit (Beyotime Biotechnology, Nantong, China).

Sun S., Guo W., Yang J.S., Qiu M., Zhu X.J, & Dai Z.M. (2018). TT(N)mGCCTC inhibits archaeal family B DNA polymerases. Scientific Reports, 8, 1990.

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Tal Gene Detection in Xoo

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For tal gene detection, Xoo genomic DNA was extracted, digested with BamHI, separated in agarose gels, and transferred to membranes for blotting as previously reported [33] (link), [44] (link). The probe was made from a DNA fragment labeled with DIG containing the repetitive region of pthXo1 (GenBank accession number: AY495676). Bacterial Genomic DNA Miniprep Kit was purchased from Axygen (USA). Restriction endonucleases and DNA molecular weight markers were provided by TaKaRa Bio (Japan). DIG-labeled Southern Blot kits were purchased from Roche (Switzerland) and Immobilon-Ny⁺ membranes were supplied by Millipore (USA).

Xu Z., Xu X., Wang Y., Liu L., Li Y., Yang Y., Liu L., Zou L, & Chen G. (2022). A varied AvrXa23-like TALE enables the bacterial blight pathogen to avoid being trapped by Xa23 resistance gene in rice. Journal of Advanced Research, 42, 263-272.

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Detecting pKEF9 Integration in S. islandicus

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Southern hybridization was employed to determine the pKEF9 integration sites in S. islandicus and also to establish the presence of the pKEF9-matching spacer 44, CRISPR locus 2, in the recipient host. A standard procedure was used (29 ) in which about 4 µg total DNA from each sample was digested with EcoRI, fractionated in 1.0% agarose gels and transferred to IMMOBILON-NY+ membranes (Millipore, MA, USA) by capillary transfer. Membrane-bound DNA was then auto-crosslinked using a UV Cross-linker (Stratagene, CA, USA). Hybridization probes were amplified by PCR (Supplementary Table S1), purified and labelled with Digoxigenin Labelling kit (Roche, Basel, Switzerland). Hybridization was performed overnight at 42°C and hybridization signals were detected using a DIG detection kit with the CDP-star (Roche) and recorded on CP-BUl X-ray films (AGFA, Mortsel, Belgium). Integration of pKEF9 at two tRNA^Glu (CTC and CTT) genes was established by PCR amplification using listed primers (Supplementary Table S1).

Liu G., She Q, & Garrett R.A. (2016). Diverse CRISPR-Cas responses and dramatic cellular DNA changes and cell death in pKEF9-conjugated Sulfolobus species. Nucleic Acids Research, 44(9), 4233-4242.

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Northern Blot Analysis of Yeast Heat Shock Genes

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Total RNA was isolated from exponentially-growing cells in SD medium at 25 °C by the method of Collart and Oliviero [11 ]. Aliquots (10 μg) of total RNA were separated by electrophoresis on 1 · 2 % (w/v) agarose gels containing formaldehyde, transferred to IMMOBILON-NY+ membranes (Millipore) and hybridized as described by the manufacturer. The 3 · 0 kbp BamHI fragment of clone pYSGal104 (courtesy of Dr. Susan Lindquist) was used as DNA probe to detect HSP104 transcripts. Gene-specific DNA probes for HSP82, SSA3, HSP26, HSP12, and ACT1 were amplified by PCR. Primer pairs used during PCR were: FSHSP82 and RSHSP82 for HSP82; fc-ssa3 and rc-ssa3 for SSA3; HSP26-F and HSP26-R for HSP26; HSP12-F and HSP12-R for HSP12; ACT1-1 and ACT1-2 for ACT1 (Additional file 1: Table S5). Estimation of band intensities of autoradiograms was performed by image analysis with NIH Image 1.62 software. Data was normalized to account for differences between samples in actual total-RNA loading.

Pérez-Landero S., Sandoval-Motta S., Martínez-Anaya C., Yang R., Folch-Mallol J.L., Martínez L.M., Ventura L., Guillén-Navarro K., Aldana-González M, & Nieto-Sotelo J. (2015). Complex regulation of Hsf1-Skn7 activities by the catalytic subunits of PKA in Saccharomyces cerevisiae: experimental and computational evidences. BMC Systems Biology, 9, 42.

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