Specific regions of the gene encoding the 16S rRNA gene were amplified using the primers U968f GC and L1401r (Heuer and Smalla, 1997 ) in a solution containing 1X PCR buffer, 0.2 mM dNTPs, 2.5 mM MgCl2, 2.5 U of recombinant Taq DNA polymerase (Promega, Madison, WI, USA), 10–40 ng of total DNA, 200 μmol of each primer and sterile Milli-Q water in a final volume of 50 μl. The reaction was performed in a thermocycler (Mastercycler Gradient, Eppendorf, Hamburg, Germany) with the following conditions: initial denaturing at 94°C for 3 min; 35 cycles at 94°C for 1 min, 55°C for 1 min and 72°C for 1 min; and a final extension at 72°C for 10 min. The denaturing gradient gel elecrtophoresis (DGGE) gels (45–65% urea and formamide) were prepared with a solution of polyacrylamide (6%) in Tris-acetate (pH 8.3). Electrophoresis was performed in Tris-acetate-EDTA buffer at 60°C at a constant voltage of 75 V for 16 h. The DGGE gels were stained with SYBR Green (Invitrogen, Carlsbad, CA, USA) and visualized using a Storm 860 Imaging System (GE Healthcare, Milwaukee, WI, USA).
Storm 860 imaging system
Manufactured by GE Healthcare
Sourced in United States
The Storm 860 Imaging System is a lab equipment product designed for image capture and analysis. It provides high-resolution imaging capabilities for various applications. The core function of the Storm 860 is to enable researchers and scientists to capture, process, and analyze digital images accurately and efficiently.
Automatically generated - may contain errors
Lab products found in correlation
Ecl plus, by Thermo Fisher Scientific (3 mentions)
Sybr green, by Thermo Fisher Scientific (2 mentions)
Recombinant taq dna polymerase, by Promega (1 mentions)
Mastercycler gradient, by Eppendorf (1 mentions)
Gapdh antibody, by Merck Group (1 mentions)
Coomassie plus bradford assay, by Thermo Fisher Scientific (1 mentions)
Dcode system, by Bio-Rad (1 mentions)
100 bp dna ladder, by Thermo Fisher Scientific (1 mentions)
Sybr gold, by Thermo Fisher Scientific (1 mentions)
Ab62461, by Abcam (1 mentions)
Ab51560, by Abcam (1 mentions)
Sc 8432, by Santa Cruz Biotechnology (1 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
Myimageanalysis software, by Thermo Fisher Scientific (1 mentions)
Ab230158, by Abcam (1 mentions)
Secondary fluorescent antibodies, by Abcam (1 mentions)
Ab283655, by Abcam (1 mentions)
Ab76067, by Abcam (1 mentions)
Total protein extraction kit, by Merck Group (1 mentions)
Sc 47724, by Santa Cruz Biotechnology (1 mentions)
7 protocols using storm 860 imaging system
1
Amplifying 16S rRNA Gene Regions for DGGE Analysis
2
Utrophin Expression in mdx Muscles
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Western blots were performed on whole muscle lysates as previously described [26 (link)]. Briefly, the gastrocnemius muscles treated mdx4cv muscles and contralateral controls (n = 4) were ground in liquid N2 and homogenized in extract buffer (50 mM Tris-HCl, 150 mM NaCl, 0.2% SDS, 24 mM Na deoxycholate, 1% NP40, 47.6 mM Na Fluoride, 200 mM Na Orthovanadate, Roche, Basel, CH). Protein concentration of whole muscle was determined by Coomassie Plus Bradford Assay (Thermoscientific, Rockford, IL). Equal amounts of protein (20 μg) were resolved on a 4–12% SDS polyacrylamide gel. The blots were incubated in N-terminal anti-utrophin (1:1000; kind gift from Stanley C. Froehner) overnight at 4°C. The GAPDH antibody (1:50,000; Sigma, St. Louis, MO) was used as a loading control as its expression was unchanged when comparing the treated and untreated mdx4cv muscles. The primary antibodies were detected with IgG HRP secondary antibodies (1:25,000; Jackson ImmunoResearch Labs). The blots were developed with ECL plus (Thermoscientific, Rockford, IL) and scanned with the Storm 860 imaging system (GE Healthcare Lifesciences, Piscataway, NJ). The band intensity was measured using Image J software (NIH).
3
PCR-DGGE Analysis of Bacterial Communities
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PCR/DGGE experiments were performed with Bacteria universal set of primers U968f-GC1-L1401 (Heuer and Smalla, 1997 ). Before DGGE analysis, the presence of PCR products was confirmed by electrophoresis in a 1.2% agarose gel run at 80 V in Tris-Borate- EDTA buffer. The gel was stained with 0.5 μg/mL ethidium bromide for 15 min then it was examined under short-wavelength ultraviolet light. A 100 bp DNA ladder (Fermentas, Lithuania) served as the molecular size standard. DGGE of the amplified gene sequences was performed using a DCode System (universal mutation detection system; Bio-Rad). The gel contained 6% acrylamide with a gradient of 45% to 65% denaturant (urea and formamide). All gels were loaded with DNA markers in the first and last lanes surrounding the lanes with samples to allow gel standardization according to the manufacturer’s instructions.
Electrophoresis was performed in 1X Tris-acetate-EDTA buffer at 60°C at a constant voltage of 75 V for 16 h. Then, the gels were stained with Sybr Gold (Invitrogen) and visualized using Storm 860 Imaging System (GE Healthcare). The results were presented as dendrograms constructed after image capture and analyzed by Pearson correlation coefficients (r). The cluster analysis was performed by the unweighted pair group method with average linkages (UPGMA) using BioNumerics software (Applied Maths, Belgium).
Electrophoresis was performed in 1X Tris-acetate-EDTA buffer at 60°C at a constant voltage of 75 V for 16 h. Then, the gels were stained with Sybr Gold (Invitrogen) and visualized using Storm 860 Imaging System (GE Healthcare). The results were presented as dendrograms constructed after image capture and analyzed by Pearson correlation coefficients (r). The cluster analysis was performed by the unweighted pair group method with average linkages (UPGMA) using BioNumerics software (Applied Maths, Belgium).
4
Gastrocnemius Muscle Protein Expression
5
Denaturing Gradient Gel Electrophoresis for Microbial Community Analysis
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DGGE was performed using a DCode system universal mutation detection system (Bio-Rad, Richmond, USA). The amplicons were applied directly to the gel containing 8% (w/v) polyacrylamide and 0.5 x TAE (20 mM Tris-acetate [pH 7.4], 10 mM sodium acetate, 0.5 mM EDTA) with a gradient of 50–65% denaturant (urea and formamide). The electrophoresis run was for 16 h at 60°C and 75 V. After electrophoresis, the gel was stained for 30 min with SYBR Green (Molecular Probes, Oregon, USA) and scanned with STORM™ 860 Imaging System (GE Healthcare, Milwaukee, USA). The cluster analysis and Pearson correlation coefficients (r) were performed by the unweighted pair group method with average linkages (UPGMA) using BioNumerics Software (Applied Maths, Belgium). The DGGE band profiles were also converted into data matrices using the Bionumerics v6.0 package. To analyze the differences between profiles and composition of bacterial communities, matrices were ordered by non-metric dimensional scaling (NMDS) [36 ,37 ] using a Bray–Curtis distance matrix with Past 3.x Software [38 (link)]. To assess the variation between different samples (A, B and Control group), a permutational multivariate analysis of variance (PERMANOVA) [39 (link)] was performed using Past 3.x Software [38 (link)].
6
Quantification of Muscle Nitric Oxide Synthases
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Another cohort of WT and db/db mice treated with and without pGz was anesthetized (100 mg/kg of ketamine and 5 mg/kg of xylazine) and sacrificed by cervical dislocation. Gastrocnemius muscles were dissected, minced, and homogenized using a total protein extraction kit (Sigma Millipore, Saint Louis, MA, USA). Total protein concentrations were determined using the bicinchoninic acid (BCA) method (Thermo-Scientific, Waltham, MA, USA). Denatured, SDS-gel separated, and membrane-immobilized proteins were incubated overnight at 4 °C with primary antibodies: anti-eNOS, dilution 1:2500 (ab300072; Abcam, Waltham, MA, USA); anti-p-eNOS, dilution 1:2500 (ab230158, Abcam, MA, USA); anti-nNOS, dilution 1:2000 (ab76067, Abcam, MA, USA); anti-iNOS, dilution 1:2500 (ab283655, Abcam, MA, USA); anti-GAPDH, dilution of 1:5000 (SC47724; Santa Cruz, CA, USA); and secondary fluorescent antibodies (Abcam, MA, USA). The resolved bands were detected with a Storm 860 Imaging System (GE Bio-Sciences, Piscataway, NJ, USA). Protein levels were quantified using myImageAnalysis software V1.0 (Thermo-Fisher Scientific, Waltham, MA, USA) and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
7
Western Blot Analysis of ER Stress Markers
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