To analyze newly synthesized RNA, cells were pulse‐labeled for 2 h with 1.2 μCi/ml of [3H]‐uridine (PerkinElmer) and then chased in non‐radioactive media for 4 h before TRIzol RNA extraction as described above. 2 μg of total RNA were resolved either on a formaldehyde‐containing 1.2% agarose gel for 18S and 28S rRNAs or on a TBE‐urea 10% polyacrylamide gel for 5S rRNA, 5.8S rRNA, or tRNAs and transferred to Hybond N+ membrane (GE Healthcare). After ultraviolet cross‐linking, the membranes were sprayed with EN3HANCE (PerkinElmer) and exposed to Kodak BioMax MS film (Kodak) at −80°C for 1 week.
Biomax ms film
Manufactured by Kodak
Sourced in United States
BioMax MS film is a high-performance laboratory film used for the detection and analysis of biomolecules. It is designed for use in various imaging applications, including Western blotting, Northern blotting, and other gel-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Hybond n membrane, by GE Healthcare (4 mentions)
β actin, by Lab Vision (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
En3hance, by PerkinElmer (2 mentions)
3h uridine, by PerkinElmer (2 mentions)
Ecl reagent, by Cytiva (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Total rna purification system, by Thermo Fisher Scientific (2 mentions)
3h adomet, by PerkinElmer (2 mentions)
Enhanced chemiluminescence kit, by Cytiva (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
35s methionine, by Cytiva (1 mentions)
Amplify solution, by Cytiva (1 mentions)
Tnt t7 coupled reticulocyte lysate system, by Promega (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cytiva (1 mentions)
Primary antibodies for hif 1α, by Novus Biologicals (1 mentions)
Biomax transcreen he, by Kodak (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Quick gdna midiprep kit, by Zymo Research (1 mentions)
Bis tris sds page, by Thermo Fisher Scientific (1 mentions)
42 protocols using biomax ms film
1
Pulse-Chase RNA Labeling and Detection
2
Fusion Protein Expression via TNT
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For the generation of fusion proteins, the TNT T7 Coupled Reticulocyte Lysate System (Promega, Mannheim, Germany) was used together with 35S‐methionine (Amersham, Freiburg, Germany) following the manufacturer's protocol. For this purpose, 1 μg of each construct and 0.5 μg of control plasmid MKKS‐pGBKT7 (encoding a C‐myc‐tagged MKKS protein of 62 kD) were used. After completed TNT reaction, 20 μL of 6 × SDS‐PAGE sample buffer was added to 5 μL of the samples, boiled for 5 min and separated by SDS‐PAGE on a 12% acrylamid gel. The gel was treated with fixation buffer and Amplify Solution (Amersham) and exposed overnight at room temperature using Kodak BioMax MS film (Kodak, Rochester, NY, USA).
3
Labeling Newly Synthesized RNA
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To label newly synthesized RNA, cells were incubated for 2 hours with [3H]-uridine (1.2 μCi/ml; PerkinElmer). Cells were counted, and the total RNA corresponding to an equal number of cells for each condition was separated by Northern blot having for the time = 0 1 μg of total RNA. Agarose-resolved RNAs were transferred to Hybond N+ membrane (GE Healthcare). After ultraviolet cross-linking, the membrane was sprayed with EN3HANCE (PerkinElmer) and exposed to Kodak BioMax MS film (Kodak) at −80°C for 1 week (22 (link)). Nascent rRNA was detected by autoradiography.
4
Hypoxia Regulates Nuclear HIF-1α
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Cultured BM-MPCs were placed in hyperoxia or hypoxia for 24 h. Nuclear protein was obtained using Pierce Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL) according to the manufacturer’s instructions. Protein content was standardized using the BCA Protein Assay kit (Pierce Biotechnology). Nuclear protein (20 μg) was separated by SDS-PAGE on 7.5% gels, transferred to polyvinylidene difluoride membranes, and blocked with 5% milk in tris-buffered saline with Tween. Protein detection was performed with primary antibodies for HIF-1α (Novus Biologicals, Littleton, CO), with β-actin serving as an internal loading control (Lab Vision, Fremont, CA). Blots were subsequently incubated with a horseradish peroxidase–conjugated secondary antibody (Amersham Biosciences, Piscataway, NJ). Blots were developed with ECL reagent (Amersham Biosciences) and exposed for 1–10 min on Kodak Biomax-MS film. Band densities were quantified with ImageJ software (National Institutes of Health).
5
Gene Expression Profiling of Human Cancer Samples
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RNA was isolated according to TRI reagent® protocol (Sigma-Aldrich, USA) and then frozen at −80°C for further preparation. The quality of RNA samples was assessed by gel electrophoresis, and quantitative analysis was performed using a GeneQuant spectrophotometer (Pharmacia, Sweden). Absorption was measured at a 260-nm wavelength, and 1 OD was equivalent to 40 μg of RNA. Reverse transcription of RNA and cDNA labeling were performed with the BD RiboQuant™ RPA system (BD Bioscences-Pharmingen, San Jose, CA, USA) and CDS Primer Mix (BD Biosciences, Clontech, Palo Alto, CA, USA). PCR reaction was then performed using the BD Atlas™ NucleoSpin® extraction kit. In order to perform gene expression profiling, Atlas Human Cancer 1.2 array membranes were used (cDNA Expression Array PT3547-3E; BD Biosciences, Clontech; cat. no. 7851-1). These macroarray membranes contain 1,176 hybridization points for various genes, of which 31 are specific for ECM proteins. Hybridization reactions were performed according to the kit manual, and then membranes were transferred to the exposure cassette BioMax TranScreen HE with Kodak BioMax MS film (Kodak, USA). Exposure times varied and depended on the isotopic activity of the cDNA (between 4 and 7 days). Film development was performed with Kodak liquid developers (Kodak). Results were digitally read with AtlasImage™ software (BD Biosciences, Clontech).
6
Analyzing PRMT5 Methyltransferase Activity
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The methyltransferase activity of PRMT5 recombinant protein was analyzed as previously described (Pei et al., 2007) with minor modifications. Briefly, purified recombinant His-PRMT5 protein was treated with 0.2 mM GSH, GSNO or not treated for 30 min and then incubated with histones, myelin basic protein (MBP) or GST-LSM4 and methyl donor S-adenosyl-L-[methyl-3 H] Met (Amersham Biosciences) in HMT buffer (20 mM Tris-HCl, pH 8.0, 4 mM EDTA, 1 mM PMSF, 0.5 mM DTT) for 2 hr at 30 C. Proteins were then subjected to SDS-PAGE separation and Coomassie bright blue staining for visualization. The SDS-PAGE gels were then treated with Amplifier (Amersham Biosciences) for 15 min for signal enhancement, dried and exposed to Kodak Biomax MS film for 2-7 days at À80 C.
7
Genomic DNA Extraction and Southern Blot
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Genomic DNA was extracted with the Quick-gDNA MidiPrep kit (ZYMO RESEARCH). Note that 4 μg of gDNA was digested over night with the EcoNI and XbaI (Landing Pad integration test) or with AseI (circuit integration test), separated on 0.8% agarose gel, transferred to a nylon membrane and probed with the indicated 32P-radiolabeled probe (Supplementary Text 1). Biomax MS film (KODAK) were stored 2 days in an exposition cassette with the membrane and revealed with a darkroom X-Ray processor (Velopex MD2000).
8
Quantifying Transgenic Seed CV-N Transcripts
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Transgenic T3 seeds harvested 2, 4, 6 and 8 weeks after pollination were analysed for the presence of CV‐N primary transcripts. Total RNA from each immature seed (200 mg) was isolated using a total RNA Purification System® (Invitrogen, Life Technologies, Grand Island, NY) according to the manufacturer's protocol. Genomic DNA was eliminated by sample digestion with 2 U of DNase 1® (Ambion, Life Technologies, Grand Island, NY) for 10 min at 37 °C. Two 32P‐labelled probes (at 106 c.p.m./mL) were used to detect primary transcripts of the cv‐n gene and the internal control corresponding to the endogenous elongation factor gene. Both probes, with 318 and 560 bp, respectively, were obtained by PCR as previously outlined (Li et al., 2006 ). Autoradiograms were obtained by exposing the membranes to BioMax MS film (Kodak, Rochester, NY).
9
MLL1-Mediated Histone H3 Methylation Assay
10
Dectin-1 Immunoblotting: Optimized Protocols
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Immunoblotting for Dectin-1 was performed using an equal protein 30 μg of cell lysate or recombinant protein per lane. Samples were loaded on a 4%–12% precast NuPAGE Bis-Tris polyacrylamide gel (Invitrogen).55–58 (link) Proteins were electrophoresed using MES-SDS buffer at 200 V for 35–45 minutes per the manufacturer's instructions (Invitrogen). Native gel electrophoresis was performed using a Novex 4% Tris-glycine gel (Invitrogen). Samples were mixed with a cold native gel sample buffer (Invitrogen) before loading. Electrophoresis was run at room temperature at a constant voltage of 80 V for 5 hours. Proteins were then transferred from the gel to a nitrocellulose membrane using the XCell II Mini-Cell and sandwich blot module (Invitrogen) in Tricine-10% methanol-0.01% SDS transfer buffer (Invitrogen) at 30 V for 1 hour. Blots were blocked for 1 hour at room temperature with 10% nonfat milk and then incubated with primary Dectin-1 antibody (R&D system) (1:500 in 1% nonfat milk) overnight at 4°C. Blots were then incubated with 1:5000 goat anti-rabbit IgG-horseradish peroxidase for 2 hours at room temperature. Signal was detected using the enhanced chemiluminescence kit (Amersham), and blots were exposed to Kodak Biomax MS film.
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