RNA was prepared by lysing 10 million cells in 2 mL of proteinase K solution (500 μg/mL proteinase K [QIAGEN]; 200 mM NaCl [Fisher Scientific]; 200 mM Tris-HCL, pH 7.5 [Wisent]; 1.5 mM MgCl2 [Fisher Scientific]; and 2% SDS [Roche]) for 30 min at 55°C. mRNA was then isolated with 60 mg of oligo(dT) (Sigma-Aldrich) using microcentrifuge spin columns (Ambion). Concentrated mRNA was quantified by absorbance at 260 nm on a NanoDrop 2000c (Thermo Scientific), and quality was verified on an agarose gel by controlling residual 28S/18S contamination. 3 μg of mRNA were separated on a 1% agarose gel containing MOPS (Laboratoire MAT) and 0.62 M formaldehyde (Sigma-Aldrich) and transferred to Immobilon-NY+ nylon membrane (Millipore) by downward alkaline blotting and hybridized with human 32P-labeled DMPK or GAPDH oligonucleotide probes. Probes were generated by random priming (New England Biolabs) of 100 ng of DMPK cDNA fragments (BglII-SacI) or GAPDH cDNA PCR-amplified fragments (Table S3 ). Overnight hybridization was conducted at 65°C in hybridization buffer (1X SSPE [Sigma]; 2X Denhardt’s [Sigma]; 10% dextran sulfate [Fisher Scientific]; 1% SDS [Roche]; 100 μg/mL salmon sperm DNA [Ambion]; and probe 1 Mcpm/mL). Relative quantification of mRNA levels on scanned autoradiograms (600 dpi) was determined by densitometry using ImageJ 1.47 software.
Immobilon ny nylon membrane
Manufactured by Merck Group
Immobilon-Ny+ nylon membrane is a laboratory product designed for use in various scientific applications. It is a nylon-based membrane that can be used for the immobilization and transfer of biomolecules, such as proteins and nucleic acids, during experimental procedures. The membrane provides a stable and efficient platform for these applications.
Automatically generated - may contain errors
Lab products found in correlation
Bio dot sf apparatus, by Bio-Rad (2 mentions)
Imagequant las 4000 biomolecular imager, by GE Healthcare (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions)
Oligo dt, by Merck Group (1 mentions)
Salmon sperm dna, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Roche (1 mentions)
Denhardt s, by Merck Group (1 mentions)
Mgcl2, by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Proteinase k, by Qiagen (1 mentions)
Chemiluminescent nucleic acid detection module kit, by Thermo Fisher Scientific (1 mentions)
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (1 mentions)
Dig high prime dna labeling and detection starter kit 2, by Roche (1 mentions)
5 protocols using immobilon ny nylon membrane
1
Quantitative Analysis of DMPK and GAPDH mRNA Levels
2
Characterization of NF-YA Transcription Factor
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EMSA was performed using the LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific). Briefly, the nuclear protein lysate from NF‐YA overexpressed SiHa and C33A cells was incubated at room temperature in 10 mM Tris‐HCl, 50 mM KCl, 2.5% (vol/vol) glycerol, 5 mM MgCl2, 50 ng/μL poly (dI‐dC) and 0.05% (vol/vol) NP‐40 binding buffer at pH 7.5. After 10 minutes, 20 fmol of the biotin‐labelled DNA fragment containing NF‐YA binding sites (CCAAT/ATTGG) was added to the equilibrated, unclear protein and then incubated at room temperature for 20 minutes. The samples were resolved on a 5% non‐denaturing polyacrylamide gel prepared in 45 mM Tris‐borate and 1mM EDTA (TBE) buffer. The specimens were electrotransferred onto a 0.45‐μm Immobilon‐NY + nylon membrane (Millipore) at 350 mA for 40 minutes on ice. Then, the membrane was cross‐linked using an ultraviolet cross‐linker (CL‐1000 with the shortwave UV of 254 nm). The blots were developed using a Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific).
3
Syndecan-1 Detection in Infected Cells
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Cell culture supernatants of infected or mock-treated HCE cells were vacuum filtered through Immobilon-Ny+ nylon membrane (Millipore Corporation, Billerica, MA) using Bio-Dot SF apparatus (Bio-Rad, Hercules, CA). Membranes were blotted overnight with antibody specific for Syndecan-1 (Santa Cruz Biotechnology, sc-6532) followed by species-specific secondary antibody and chemiluminescence detection using ImageQuant LAS 4000 biomolecular imager (GE Healthcare Life Sciences), as described above.
4
Syndecan-1 Detection in Infected Cells
5
Minichromosome Analysis via Southern Blot
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To examine the eliminated minichromosomes, genomic DNA was extracted from vegetative cells, conjugating and re-fed cells at different time points. For standard gel electrophoresis, total DNAs were extracted from cells at different time points as previously described [61 (link)]. Uncut or HindIII-digested DNA were separated in a 0.6% agarose gel. After electrophoresis, DNA was transferred to a piece of IMMOBILON-NY+ nylon membrane (Millipore, Bedford, MA). The digoxigenin-labeled probes were generated using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Indianapolis, IN) and mixed with the membrane for hybridization at 42°C overnight. The membranes were washed in 2× saline-sodium citrate (SSC) with 0.1% SDS twice and 0.5× SSC with 0.1% SDS at 65°C twice before detection of the luminescence using x-ray films.
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