In accordance with the instructions provided for the MAXIscript In Vitro Transcription Kit (Ambion), cDNA fragments containing chimeric junctions for probe synthesis were tagged with SP6- (antisense strand) and T7- (sense strand) targeting sequences by PCR. SP6 or T7 polymerase was added to generate 32P-UTP labeled antisense transcripts or non-labeled sense transcripts, respectively. The 32P-UTP labeled antisense transcripts were used as protective probes in RNase protection assay (RPA). RPA was performed using the RPA III kit (Ambion). In brief, 10 μg total RNA and 100 ng antisense probes were hybridized at 56°C for 16 h, and non-hybridized fragments were then digested by RNase A and T1. The protected fragments were separated on a 5% denaturing (8 M Urea) polyacrylamide gel, and signals were developed by exposure on X-OMAT blue films (Kodak).
X omat blue film
Manufactured by Kodak
Sourced in United States
X-OMAT blue films are a type of photographic film used in medical and industrial imaging applications. They are designed to provide high-quality, consistent results for a variety of X-ray and other imaging procedures. The films are sensitive to specific wavelengths of light, allowing for accurate and reliable image capture.
Automatically generated - may contain errors
Lab products found in correlation
Rpa 3 kit, by Thermo Fisher Scientific (1 mentions)
Maxiscript in vitro transcription kit, by Thermo Fisher Scientific (1 mentions)
Mouse anti gfap, by Merck Group (1 mentions)
Rabbit anti jak1, by Santa Cruz Biotechnology (1 mentions)
Mouse anti stat3, by BD (1 mentions)
Mouse anti tuj1, by Fortrea (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
4 protocols using x omat blue film
1
RNase Protection Assay for Gene Expression
2
Western Blot Analysis of JAK-STAT Pathway
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The antibodies used for western were as follows: mouse anti-Gfap (Sigma), rabbit anti-Jak1 (Santa Cruz Biotechnology, Dallas, TX), rabbit anti-Il6st (Santa Cruz, C-20), rabbit anti-Lifr-beta (Santa Cruz, C-19), rabbit anti-Stat1 (BD Biosciences, San Jose, CA), mouse anti-Stat3 (BD Biosciences), mouse anti-phosphotyrosine Stat1 (BD Biosciences), rabbit anti-phosphotyrosine Stat3 (Cell Signaling, Danvers, MA), mouse anti-TuJ1 (Covance, Princeton, NJ) and mouse anti-β-actin (Actb) (Sigma). Secondary goat anti-mouse or anti-rabbit IgG-horseradish antibodies (Calbiochem, EMD Millipore, Billerica, MA) were used, and detection was performed using the ECL plus chemiluminescence (PerkinElmer, Waltham, MA) on X-Omat Blue films (Kodak).
3
GTPBP4 Protein Expression Analysis
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Harvested the cells when the cell fusion rate reached 70–80%, aspirated the cells of different transfection groups, and placed them in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris -HCl pH 8.0). After resuspension, added protease inhibitor cocktail (Sigma, USA). Used the Bradford reagent (BioRad, NY) to quantify the protein. Added 25 μg of protein to 10% SDS-PAGE (BioRad) gel wells and performed electrophoresis. After electrophoresis, transferred the protein to a polyvinylidene fluoride membrane. Cultured the nonspecific binding sites in 5% skimmed milk powder PBS for 1 hour to block and then washed with 0.1% Tween-20. Diluted GTPBP4 by 1 : 3000 (EPR3500, Abcam, USA) and then placed it on the membrane for incubation overnight. After washing, incubated the membrane with 1 : 5000 diluted anti-rabbit secondary antibody (conjugated with horseradish peroxidase). Finally, added Western blotting luminescence reagent and imaged in Kodak X-Omat blue film.
4
FOXL1 Protein Expression Analysis
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Total proteins were extracted from NOZ cells 48 h after transfection with pcDNA-FOXL1 or pcDNA3.1. The protein content was quantified by Bradford protein assay. 40 µg of protein was separated by 10–12% SDS-PAGE and electrotransferred to PVDF membranes (Millipore, Billerica, MA, USA). The membrane was blocked with 5% non-fat dry milk, and probed with corresponding primary antibodies (Santa Cruz Bio) overnight at 4°C, followed by incubation with horse radish peroxidase-coupled secondary antibodies (Santa Cruz Bio). Horseradish peroxidase activity was visualized using enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL, USA) and exposed to X-OMAT-Blue film (Kodak, Rochester, NY, USA).
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