Hybond nylon membrane

Manufactured by GE Healthcare

Sourced in United States, United Kingdom

Hybond nylon membrane is a laboratory product designed for use in various molecular biology techniques. It is a nylon-based membrane that can be used for DNA and RNA transfer, immobilization, and detection applications. The membrane provides a stable and efficient platform for these processes, facilitating accurate and reliable results.

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Lab products found in correlation

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Close spelling variants of this product

Hybond-NX nylon membrane Hybond-XL nylon membrane Amersham Hybond-NX nylon membrane Hybond-A nylon membrane Hybond-N+ positively charged nylon membranes Hybond-N + nylon transfer membrane Amersham Hybond nylon membrane Hybond NX neutral nylon membrane Hybond-N+ nylon membrane Amersham Hybond-N+ nylon membrane

30 protocols using hybond nylon membrane

Chloroplast Genomic Analysis by Blotting

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Total DNA from wild-type and transplastomic plants was extracted by a cetyltrimethylammonium bromide (CTAB)-based method (2% CTAB, 1.4 mM NaCl, 0.1 mM Tris–HCl pH 8.0, 20 mM EDTA pH 8.0; Doyle and Doyle, 1990 ). Total RNA was isolated using the RNAiso plus reagent following the manufacturer’s instruction (Takara, Japan). For Southern blot analyses, samples of 5 µg total DNA were digested with the restriction enzymes AgeI and MluI for 12–16 h, separated by electrophoresis in 1% agarose gels, and transferred onto Hybond nylon membranes (GE Healthcare) by capillary blotting. A PCR product covering a portion of the psbZ coding region (You et al., 2019 (link)) was used as a hybridization probe.
For RNA gel blot analyses, RNA samples were denatured, separated in formaldehyde-containing 1% agarose gels and blotted onto Hybond nylon membranes (GE Healthcare). A PCR product generated by amplification of ACT cDNA with primer pair act-N-F/act-N-R served as probe to determine dsRNA amounts. The expression of potato plastid-encoded psbA gene served as internal control. The psbA gene-specific probe was synthesized using primer pair psbA-N-F/psbA-N-R (see Supplementary Table S2). The probe was labeled with the DIG-High Prime DNA Labeling and Detection Starter Kit II following the manufacturer’s instructions (Roche, USA).

He W., Xu W., Xu L., Fu K., Guo W., Bock R, & Zhang J. (2020). Length-dependent accumulation of double-stranded RNAs in plastids affects RNA interference efficiency in the Colorado potato beetle. Journal of Experimental Botany, 71(9), 2670-2677.

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Radioactive DNA Labeling and Northern Blot

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The DNA labeling reaction mixture contained a buffer (50 mM Tris–HCl pH 7.6, 10 mM MgCl₂, 5 mM of DTT, 0.1 mM spermidine), 10 U T4 polynucleotide kinase, 20 mCi of (γ-³²P)ATP with an activity of 5000 Ci/mmol (Hartmann Analytic, Braunschweig, Germany) and 20 pmol of a DNA oligomer (Table S1). After an hour-long incubation at 37 °C, the ³²P labelled products were purified using a G25 column (Sigma-Aldrich, St. Louis, MO, USA). The radioactivity level of the labeled molecules was measured using a scintillation counter. The total RNA isolated from the different cultures was separated on 12% (w/v) polyacrylamide gels, electrotransferred onto Hybond-Nylon membranes (GE Healthcare, Chicago, IL, USA), crosslinked with UV light (120 mJ/cm²) and prehybridized in a 2xSSC, 1X Denhard solution for 1 h at 37 °C. Hybridization of ³²P labelled DNA probes (20 × 10⁶ cpm) was carried out at 42 °C overnight in PerfectHyb^TM Plus solution (Sigma). Then, the hybridization mixture was discarded, and the blot was washed in the same solution several times, until the radioactivity in the solution disappeared. Finally, the blots were analyzed using phosphor imaging screens and a FLA-5100 image analyzer with MultiGauge software (FujiFilm).

Kazimierczyk M., Wojnicka M., Biała E., Żydowicz-Machtel P., Imiołczyk B., Ostrowski T., Kurzyńska-Kokorniak A, & Wrzesinski J. (2022). Characteristics of Transfer RNA-Derived Fragments Expressed during Human Renal Cell Development: The Role of Dicer in tRF Biogenesis. International Journal of Molecular Sciences, 23(7), 3644.

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Quantitative Western Blotting for PDGF-B

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Laboratory investigations including complete blood count using SYSMEX KX21N (Japan), biochemical analysis using Cobas c311 (Germany) for kidney function, liver function, and fasting blood glucose, coagulation profile using Stago STA (France), and DD using Immulite 1000 (USA).
Then, protein expression of PDGF-B is analyzed using the Western blotting technique as illustrated in Figure 1.
Plasma samples were fractionated through 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Equal amounts of protein (30 μg) were separated and transferred onto Hybond™ nylon membranes (GE Healthcare, Buckinghamshire, UK). Immunoblots were probed with the primary antibody; anti-PDGF-B (ab23914 Abcam, Cambridge, UK) at 4°C overnight. The membranes were then washed and incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized using a gel documentation system (GelDoc-it, UVP, England) and Total lab analysis software (CLIQS, liverpool, UK), www.totallab.com (Ver. 1.0.1).

Alhabibi A.M., Eldewi D.M., Wahab M.A., Farouk N., El-Hagrasy H.A, & Saleh O.I. (2019). Platelet-derived growth factor-beta as a new marker of deep venous thrombosis. Journal of Research in Medical Sciences : The Official Journal of Isfahan University of Medical Sciences, 24, 48.

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RNA Transfer and Crosslinking Protocol

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After the completion of gel electrophoresis, the acrylamide gels were stained with EtBr (0.05 µg/mL) for visualization using UVP Imager and/or Typhoon using EtBr filter. This was followed by pre-soaking the gels in either 1× MOPS (agarose gels) or 1× TBE buffer (acrylamide gels). RNA in the gels was transferred onto positively charged Hybond nylon membranes (GE Healthcare, Chicago, IL USA) pre-soaked in the same buffer used for equilibration. Electroblotting was performed at 80 mA constant current for 1 h for agarose gels and 18 mA for 1 h for acrylamide gels, respectively. After the completion of the transfer, membranes were placed on pre-soaked filter papers with either 1× MOPS or 1× TBE buffers and first UV crosslinked at 700 µJ. This was followed by chemical crosslinking by EDC rather than baking, as described earlier [33 (link)]. Briefly, a filter paper soaked in a freshly prepared solution of EDC was placed on a plastic wrap and the UV-cross-linked membrane was placed upside down on the soaked filter paper and wrapped properly. This was followed by incubation at 60 °C for 2 h. After chemical crosslinking, the membranes were washed prior to pre-hybridization or dried and stored at −20 °C for later use.

Ahmad W., Gull B., Baby J, & Mustafa F. (2021). A Comprehensive Analysis of Northern versus Liquid Hybridization Assays for mRNAs, Small RNAs, and miRNAs Using a Non-Radiolabeled Approach. Current Issues in Molecular Biology, 43(2), 457-484.

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Quantitative Analysis of snoRNAs

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NOP1 or nop1-ts cells were grown in yeast–peptone–dextrose at 37 °C to an OD₆₀₀ of ∼0.6. Total RNA from three biological replicates of each strain was isolated using the hot phenol method. snoRNAs were separated on 8% acrylamide/urea gels, transferred to Hybond nylon membranes (GE Healthcare), and probed as indicated. Bands were quantified using Image Lab software (Bio-Rad).

Khoshnevis S., Dreggors-Walker R.E., Marchand V., Motorin Y, & Ghalei H. (2022). Ribosomal RNA 2′-O-methylations regulate translation by impacting ribosome dynamics. Proceedings of the National Academy of Sciences of the United States of America, 119(12), e2117334119.

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EMSA Assay for STAT6 and PPARγ

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Nuclear extracts were prepared as described above. Oligonucleotides corresponding to the STAT6 (5′-TGCCTTAGTCAACTTCCCAAGAACAGA-3′) and PPARγ (5′- GGAACTAGGTCAAAGGTCATCCCCT-3′) binding site consensus sequences were synthesized and end-labeled with biotin by Invitrogen (Invitrogen, Shanghai, China). EMSAs were performed using the LightShift chemiluminescent EMSA kit (Thermo Scientific, Rockford, IL, USA). Briefly, 10 fmol of biotin-labeled, double strand probe were incubated for 20 minutes at room temperature in 20 μl of EMSA binding buffer containing 2.5% glycerol, 5 mM MgCl₂, 50 ng/μl poly (dI-dC), 0.05% Nonidet P-40, and 6 μg of nuclear proteins. For competition EMSA, 200-fold (2 pmol) excess unlabeled, double strand probe was added to the binding reaction. The DNA-nuclear protein complexes were resolved by electrophoresis in 6% nondenaturing polyacrylamide gel in 0.5 × Tris-borate-EDTA (TBE) buffer at 100 V. Gels were then electroblotted onto Hybond nylon membranes (GE Healthcare, Freiburg, Germany) at 380 mA for 50 minutes. The membranes were then cross-linked for 15 minutes with the membrane face down on a transilluminator at 312 nm, and the biotinylated protein-DNA bands were detected with HRP-conjugated streptavidin using the chemiluminescent nucleic acid detection system.

Li L., Wu Y., Wang Y., Wu J., Song L., Xian W., Yuan S., Pei L, & Shang Y. (2014). Resolvin D1 promotes the interleukin-4-induced alternative activation in BV-2 microglial cells. Journal of Neuroinflammation, 11, 72.

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RNA Transfer and Membrane Preparation

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After the completion of gel electrophoresis, the acrylamide gels were stained with EtBr (0.05 µg/mL) for visualization using UVP Imager and/or Typhoon using EtBr filter. This was followed by pre-soaking the gels in either 1× MOPS (agarose gels) or 1× TBE buffer (acrylamide gels). RNA in the gels was transferred onto positively charged Hybond nylon membranes (GE Healthcare, Chicago, IL, USA) pre-soaked in the same buffer used for equilibration. Electroblotting was performed at 80 mA constant current for 1 h for agarose gels and 18 mA for 1 h for acrylamide gels, respectively. After the completion of the transfer, membranes were placed on pre-soaked filter papers with either 1× MOPS or 1× TBE buffers and crosslinked using UV crosslinker at 700 µJ (Stratagene, San Diego, CA, USA). This was followed by baking at 80 °C for two hours. After crosslinking and baking, membranes were wrapped and stored at −20 °C. This was followed by signal detection and imaging which will be discussed commonly for both northern blotting and liquid hybridization techniques in Section 2.6.

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Plastid Transformation Analysis Protocol

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Leaf tissue frozen in liquid nitrogen was used to isolate total plant DNA by a cetyltrimethylammoniumbromide (CTAB)-based method (27 ). Total cellular RNA was extracted using the peqGOLD TriFast reagent (Peqlab GmbH). For Southern blot analysis, samples of 5 μg total DNA were digested with the restriction enzyme BglII, separated by gel electrophoresis in 0.8% agarose gels, and transferred onto Hybond nylon membranes (GE Healthcare) by capillary blotting (28 (link)). For northern blot analysis, samples of 3.5 μg total cellular RNA were electrophoresed in formaldehyde-containing 1.5% agarose gels and blotted onto Hybond nylon membranes (29 (link)). Hybridizations were performed at 65°C in Church buffer (30 (link)). Hybridization probes were purified by agarose gel electrophoresis following extraction of the DNA fragments of interest from excised gel slices using the NucleoSpin^® Extract II kit (Macherey-Nagel, Düren, Germany). A 550-bp PCR product generated by amplification of a portion of the psaB coding region (25 (link)) was used as an RFLP probe to verify plastid transformation and assess homoplasmy. Transgene-specific probes for northern blot analyses were generated by PCR using the primers listed in Supplementary Table S2.

Emadpour M., Karcher D, & Bock R. (2015). Boosting riboswitch efficiency by RNA amplification. Nucleic Acids Research, 43(10), e66.

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Detecting Viral Genome Strands in NoNRV1-ZJ

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To assess the efficacy of curing NoNRV1-ZJ from its host 9-1 by Ribavirin treatment, both positive and negative strands of the virus were detected by RNA gel blotting assays, as described previously [53 (link)]. Briefly, total RNAs were extracted from the untreated (9-1) and drug-treated (9-1C) mycelia using TRIzol (Thermo-Fisher), separated in 1.5% agarose gel containing 7% formaldehyde, and transferred onto a Hybond + nylon membrane (GE). The positive and negative strands of the viral genome were separately probed using the digoxigenin (DIG)-labeled DNA oligos, oligo-S (5′-ACAAGACGCTCATTCTGTTTGACCCCATCCCGACCCACG-3′) and oligo-AS (5′-TCTCGTGTTCGTCACAATGTCTACGACAAACGCAATCG-3′), respectively. The DIG-labeled oligos were detected using a chemiluminescence-based DIG detection kit (Roche), according to the manufacturer’s instructions.

Wang X., Lai J., Hu H., Yang J., Zang K., Zhao F., Zeng G., Liao Q., Gu Z, & Du Z. (2022). Infection of Nigrospora nonsegmented RNA Virus 1 Has Important Biological Impacts on a Fungal Host. Viruses, 14(4), 795.

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Electrophoretic Mobility Shift Assay for NigR

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EMSA was performed using a LightShift^™ EMSA Optimization & Control Kit (Thermo-Fisher Scientific, Waltham, MA) with biotin-labeled DNA probes and purified NigR according to the manufacturer’s instructions. Poly(dI-dC) was used in all reactions as a non-specific competitor at a concentration of 1 μg per reaction (Ajdic & Ferretti, 1998 (link); Nieto et al., 2001 (link); Chawla et al., 2010 (link)). Unlabeled specific competitor was added where indicated. Both non-specific and specific competitors were used as described previously (Ajdic & Ferretti, 1998 (link); Gaigalat et al., 2007 (link); Nentwich et al., 2009 (link)). An unrelated protein InvB (Type III secretion chaperone from Salmonella enterica; Lilic et al., 2006 (link)) was used as a negative control. The 20-μl binding reactions were incubated at room temperature for 20 min and the DNA–protein complex was separated from the free probe by electrophoresis on 4 or 5% native polyacrylamide gel in TBE buffer. The material was transferred to positively charged Hybond nylon membrane (GE Healthcare, Pittsburgh, PA) using Trans-Blot Semi Dry Transfer Cell (Bio-Rad) according to the manufacturer’s instructions. The membrane was cross-linked for 10 min using UV Crosslinker (UVP HL-200 HybriLinker) and developed using Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific) following the manufacturer’s instructions.

Vujanac M., Iyer V.S., Sengupta M, & Ajdic D. (2015). Regulation of Streptococcus mutans PTSBio by the transcriptional repressor NigR. Molecular oral microbiology, 30(4), 280-294.

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