The mRNA expression levels of p22phox, p47phox, gp91phox and p67phox in the renal cortical tissues were quantitatively analyzed by reverse transcription polymerase chain reaction (RT-PCR), as described previously (14 (link),15 (link)). Primer sequences for the analysis of p22phox, p47phox, p67phox, gp91phox and GAPDH mRNA are shown in Table I . GAPDH was measured as an invariant housekeeping gene for an internal control. Amplified cDNA bands were detected by Goldview staining (RuiTaibio, Beijing, China) and the quantities of mRNA were evaluated using the Gene Genius imaging system (Syngene, Cambridge, UK).
Genegenius imaging system
Manufactured by Syngene
Sourced in United Kingdom
The GeneGenius Imaging System is a versatile laboratory instrument designed for capturing high-quality images of electrophoresis gels and blots. It provides a reliable and efficient method for visualizing and documenting various biomolecular samples, including DNA, RNA, and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti actin, by Merck Group (1 mentions)
Anti phospho smad1 5 8, by Cell Signaling Technology (1 mentions)
Ecl kit, by Sartorius (1 mentions)
Human phospho rtk array kit, by R&D Systems (1 mentions)
Human phospho kinase array kit, by R&D Systems (1 mentions)
Miniamp plus thermal cycler, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence detection kit, by GE Healthcare (1 mentions)
β actin, by Merck Group (1 mentions)
Horseradish peroxidase conjugated to appropriate secondary antibodies, by Bio-Rad (1 mentions)
Thermal cycler, by Bio-Rad (1 mentions)
8 protocols using genegenius imaging system
1
Quantitative Analysis of NADPH Oxidase mRNA
2
Western Blotting Analysis of Endofin and Phospho-SMAD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were homogenised in phosphatase inhibitor lysis buffer and western blotting was carried out as previously described40 (link). Blots were incubated with antibodies: anti-endofin14 (link) (1:30000), anti-phospho-SMAD1/5/8 (1:3000; Cell Signaling Technology, Danvers MA, USA), anti-HA 12CA5 hybridoma supernatant (1:2000; ATCC, Manassas, VA, USA) and anti-actin (1:20000; Sigma-Aldrich, Missouri, USA). Bands developed on film were scanned using Scanmaker 9800 XL plus (Microtek International Inc. Hsinchu, Taiwan) and subsequently quantified by densitometry using the GeneGenius Imaging System (Syngene, Cambridge, UK).
3
Quantitative Protein Analysis by SDS-PAGE and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The SDS/PAGE and Western Blotting were performed as previously reported48 (link). For densitometry, the Gene-Genius Imaging System (Syngene, Cambridge, UK) was used. Measurement of the mean pixel density of each band was performed using the ImageJ software (NIH). Measurements were standardized to GAPDH or α-tubulin loading controls.
4
Molecular Profiling of Cellular Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 20 μg protein was loaded and separated on 10% SDS-PAGE. After electrophoresis, proteins were transferred to a PVDF membrane and blocked with 5% milk/Tris-buffered saline and Tween-20 buffer. The membranes were incubated with ZO-1 (1:1000), cytokeratin (1:1000), plasminogen activator inhibitor 1(PAI-1) (1:1000), fibronectin (1:1000), TGF-β1 (1:1000), phosphor-Smad3 (1:500) and phosphor-Smad2/3 (1:500) at 4 °C overnight. Afterwards, the membranes were washed and incubated at room temperature for 1 h with the secondary antibody and developed with an ECL kit (Biological Industries, China).The image was captured with the GeneGenius imaging system (Syngene, Cambridge, UK). Band intensity was measured with the Image J software (Bethesda, MD). All primary and second antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
5
Profiling Kinase Phosphorylation using Arrays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The effects of miR-378a-5p on phosphorylation of various protein phosphosites were determined with Human Phospho-Kinase Array Kit (Cat. no. ARY003; R&D Systems, Inc., Minneapolis, MN, USA) and Human Phospho-RTK Array Kit (Cat. no. ARY001; R&D Systems, Inc.). The arrays were performed according to the manufacturer's instructions. The intensities were quantified using GeneGenius imaging system and GeneTools software version 4.01 (SynGene, Cambridge, UK).
6
Strain Differentiation by (GTG)5-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
(GTG)5-PCR was performed to investigate whether tested isolates were the same or different strain [26 (link)]. Amplification was carried out in a 25 μL reaction mixture consisting of 50 ng/μL genomic DNA, 1 μM of the (GTG)5 primer (5′-GTG GTG GTG GTG GTG-3′ ), 12.5 μL of Blue TEMpase Hot Start 2x (VWR, Dublin, Ireland). Amplification was performed in a MiniAmp Plus Thermal Cycler (ThermoFisher Scientific, Waltham, Massachusetts, USA). The PCR conditions applied were: initial denaturation 95°C for 15 min, followed by 35 cycles of 95°C for 30 sec, annealing at 40°C for 40 sec and elongation at 72°C for 30 sec with a final extension step at 72°C for 5 min. The rep-PCR products were electrophoresed in a 1.5% (w/v) agarose gel for 3 h. Banding patterns were visualized using GeneGenius Imaging System (Syngene, Cambridge, UK) and further analysed using GelJ [27 (link)]. Dendrogram was generated using the following parameters: similarity method (Dice), linkage (UPGMA) and tolerance of 2%.
7
Western Blot Analysis of Nox4 and Smad Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells (105cells/well) were cultured in 6-well plates, and protein was extracted as previously described [29 (link)]. Primary rabbit polyclonal anti Nox4 (1:1000, kindly provided by Prof. Ajay M. Shah), pSmad2 (1;1000; Calbiochem, Merck Millipore, Billerica, MA, USA), total Smad (1: 1000; Abcam, Waterloo, NSW, Australia), and mouse monoclonal β-actin (1:4000; Sigma-Aldrich) antibodies were used. Proteins were detected with enhanced chemiluminescence detection kit (GE Healthcare, Sydney, NSW, Australia) with horseradish peroxidase conjugated to appropriate secondary antibodies (Bio-Rad). The GeneGenius Imaging System from Syngene was used to capture the images.
8
Bacterial Identification via 16S rDNA Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sequencing of 16S rDNA was performed to identify the selected isolates. DNA extraction was performed using the Quick Genomic DNA Extraction kit (DongSheng Biotech, China) . PCR amplification of 16S rDNA was performed using the universal 16S rDNA primer, with the forward sequence 5′-GCTGGCGGCATGCTTAACACAT-3′ and reverse primer sequence 5′-GGAGGTGATCCAGCCGCAGGT-3′, in a thermal cycler (Bio-Rad, USA) (Ruiz et al. 2000) . The program for amplification consisted of (i) denaturation at 95 °C for 5 min, (ii) 35 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C for 1 min, extension at 72 °C for 2 min, (iii) final extension at 72 °C for 4 min, and finally maintained at 4 °C until further use. The PCR amplicons were subjected to agarose gel electrophoresis at 100 V for 60 min. The gel was visualized using the GeneGenius Imaging System (Syngene, UK). PCR products were purified using PCR and DNA Fragment Purification Kit (DongSheng Biotech, China) and sequencing was performed by Center for Chemical Biology (Universiti Sains Malaysia, Malaysia). The nucleotide sequences of the isolates were analyzed using the BLAST program from NCBI (http://www.ncbi.nlm.nih.gov).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!