Statistical analysis was performed using GraphPad Prism® version 5.02 (GraphPad Software Inc., La Jolla, CA, USA). All data are expressed as the mean ± standard error of the mean and optical density values were determined using a gel imaging system (Alpha Innotech, San Leandro, CA, USA). Student’s t-test was used to compare the differences between two groups and statistics between groups were assessed using analysis of variance followed by Dunnett’s-test. P<0.05 was considered to indicate a statistically significant difference.
Gel imaging system
Manufactured by Bio-Techne
Sourced in United States
The Gel imaging system is a laboratory instrument designed for the visualization and analysis of electrophoresis gels, such as those used for DNA, RNA, or protein analysis. The core function of the system is to capture high-quality images of stained gels and provide tools for further data analysis.
Automatically generated - may contain errors
Lab products found in correlation
Western blotting luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Roche (1 mentions)
β actin, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Secondary antibody conjugated to horseradish peroxidase, by Beyotime (1 mentions)
Avaii, by New England Biolabs (1 mentions)
Primer star max, by Takara Bio (1 mentions)
Ecl system, by Cytiva (1 mentions)
Sc 482, by Santa Cruz Biotechnology (1 mentions)
Pab7888, by Abnova (1 mentions)
6 protocols using gel imaging system
1
Statistical Analysis of Biological Samples
2
Quantitative Protein Analysis via Western Blot
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The total protein concentration was determined using the BCA protein assay kit (Beyotime, China). The protein samples were subjected to electrophoresis via SDS-PAGE and were transferred onto a PVDF membrane (Roche, UK) using a blot system, according to standard protocols. Antibodies against GR (Santa Cruz, USA) and β-actin (Beyotime, China) and a secondary antibody conjugated to horseradish peroxidase (Beyotime, China) were used for image detection. The images were visualized by Western Blotting Luminol Reagent (Santa Cruz, USA) via Gel Imaging System (Alpha Innotech, US). The band intensity was determined using the ImageJ software. The relative ratio (GR/β-actin) was calculated.
3
Alkali-Denatured DNA Fragmentation Analysis
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Cells were trypsinised and genomic DNA isolated using the QIAprep Spin Miniprep kit. DNA concentration was determined using a NANODROP 2000 Spectrophotometer. 2.5 μg DNA was incubated with 0.3 N NaOH for 2.5 hours at 55°. Subsequently samples were loaded on alkaline agarose gels (0.7% Agarose, 50 mM NaOH, 1 mM EDTA, pH 8) and run for 18 hours at 1Volt/cm. Next, the gel was incubated for 2 hours in the neutralizing buffer (1 M Tris pH 7.6, 1.5 M NaCl). Gels were stained overnight with 0.5 M Ethidium Bromide and imaged under UV via Alpha Innotech Gel Imaging system. All the images were analysed using ImageJ in the same manner.
4
CAPS Marker Development for BnAHAS1 Mutation
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The sequence comparison of BnAHASs revealed that the nucleotide sequence 540-544 bps from the translation starting site in the WT allele of BnAHAS1 in the line ZS9 was GGTCC, which was mutated to GGTCT in the mutant line K5 (see Section 2.5 ). Therefore, the restriction endonuclease AvaII with the restriction sites GGWCC was employed to develop the CAPS marker for the detection of the causal point mutation in BnAHAS1. A pair of AS-PCR primers (BnA1F1 and BnA1R4, Table S3 ) was designed to amplify the target sequence containing the mutant locus. The PCR mixture (40 μL) contained 100 ng template DNA, 0.75 μM for each primer and 20 μL Primer Star Max (Takara, China). PCR amplification was carried out with 36 cycles at 98 °C for 10 s, 56 °C for 5 s and 72 °C for 10 s. All PCR products were digested with 10 U AvaII (New England Biolabs, Ipswich, MA, USA) for about 30 min at 37 °C at a final volume 25 μL. Subsequently, the products were separated on 2.5% agarose gel, stained with ethidium bromide and visualized using a gel imaging system (Alpha Innotech, Shanghai, China).
5
Western Blot Analysis of Sema3A and STAT3
6
SDS-PAGE Protein Purification Analysis
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To check the purification, different fractions were run into a SDS-PAGE of 12.5% (w/v) gel containing 0.1% (w/v) SDS by the method of Laemmli (1970) followed by staining with Coomassie Brilliant Blue (G-250). Molecular weight was measured using Alpha Innotech Gel Imaging System.
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