The amount of DNA recovered from each sample processed via the four methods described above (Fig 1.3, 1.8, 1.13 and 1.17 ) was quantified with the Quant-iT PicoGreen dsDNA Kit (Invitrogen). DNA integrity and fragment size in samples was determined by gel electrophoresis in a 1% (w/v) agarose (Promega) gel stained with 0.4 μg/mL ethidium bromide. A 100 bp DNA ladder (Promega) was run alongside both samples as a molecular weight marker. DNA was visualised under UV illumination using a Bio-Rad Gel Doc 1000 (Bio-Rad) and photographed using a Kodak DC90 Zoom Digital Camera (Kodak) in conjunction with the Kodak 1D v3.6 image analysis software.
Gel doc 1000
Manufactured by Bio-Rad
Sourced in United States, Italy
The Gel Doc 1000 is a compact and versatile gel documentation system designed for the analysis of DNA, RNA, and protein samples. It captures and digitizes images of gels, blots, and other samples illuminated using UV, visible, or white light. The system provides basic image analysis capabilities, allowing users to view, annotate, and quantify the captured images.
Automatically generated - may contain errors
Lab products found in correlation
Multi analyst, by Bio-Rad (5 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (4 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Molecular analyst software, by Bio-Rad (3 mentions)
Multi analyst version 1, by Bio-Rad (3 mentions)
Fibronectin, by Santa Cruz Biotechnology (3 mentions)
Ecltm prn 2106, by Cytiva (3 mentions)
Immobilon fl 0.4 μm polyvinylidene difluoride membranes, by Merck Group (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Super signal ultra chemiluminescence detection reagents, by Thermo Fisher Scientific (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Supersignal west pico chemiluminescent substrate kit, by Thermo Fisher Scientific (2 mentions)
Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (2 mentions)
Collagen 1, by Abcam (2 mentions)
α sma, by Abcam (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
β actin, by Merck Group (2 mentions)
75 protocols using gel doc 1000
1
DNA Quantification and Integrity Analysis
2
Western Blot Analysis of Vascular Proteins
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Arteries or cells were placed into RIPA lysis buffer containing protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Protein samples (30 μg) were separated with 7.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, NJ, USA). The membrane was blocked with blocking buffer (TBS, 0.1% Tween-20, 5% non-fat milk or 2% BSA) for 2 hours at room temperature and incubated with primary antibodies against eNOS (1:1000, Cell Signaling Technology, Danvers, MA, USA), p-eNOS (ser1177) (1:1000, Abcam, Cambridge, MA, USA), arginase I (1:1000, Abcam, Cambridge, MA, USA), arginase II (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), UT-B (1:1000, a kindly gift from Dr. Trinh-Trang-Tan, INSERM, Paris, France), β-actin (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Goat anti-rabbit IgG horseradish peroxidase (1:10000, Abcam, Cambridge, MA, USA) or goat anti-mouse IgG horseradish peroxidase (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were incubated for 60 minutes at room temperature the next day, respectively. The blots were developed with ECL kit (Applygen Technologies Inc, Beijing, China) and finally exposed to X-ray films. Relative protein expression levels were quantified by optical density analysis (Quantity-One software, Bio Rad Gel Doc 1000, Milan, Italy) and normalized to β-actin.
3
Western Blot Analysis of Lung Tissue
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The cryopreserved right side lung tissues were harvested and used for western blot analysis as previously described (15 (link)). i) The right rat lung tissues conserved at −70°C were homogenized by adding radioimmunoprecipitation assay lysis buffer containing phenylmethylsulfonyl fluoride. ii) Quantification was conducted using Coomassie brilliant blue following centrifugation at 256 × g; iii) Equivalent total proteins (50 mg) were obtained from each sample, and they were transferred to nitrocellulose membranes using 10% SDS-polyacrylamide gel electrophoresis (PAGE); iv) rabbit anti-rat hOGG1 (GTX20204; GeneTex, Inc., Irvine, CA, USA) and rabbit anti-rat PPARγ (ab209350; Abcam primary antibodies were diluted to 1:1,000 and incubated with the membrane overnight at 4°C; v) HRP-labeled goat anti-rabbit IgG (ab6721; 1:1,000; Abcam) secondary antibody was incubated at room temperature with the membrane for 1 h; vi) enhanced chemiluminescent substrate was added for coloration for 15 min. The integrated ODs of the bands were analyzed using ImageMaster software (v1.0.3.7) with the Bio-Rad Gel Doc1000 imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The imaging system was used to acquire images of the X-ray films and the analysis software was used to analyze and calculate the integrated ODs (13 (link),16 (link)).
4
Genomic DNA Extraction and Amplification from Parasite Specimens
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Genomic DNA from parasite specimens (small pieces of host’s infected tissue) was extracted using the Quick-DNA MicroPrep Kit (Zymo Research, USA) according to the manufacturer's instructions, preceded by an overnight digestion with proteinase K. Two markers were used for amplification and sequencing: a fragment of approximately 400 bp of the cytochrome c oxidase subunit 1 (cox1) was amplified using the primers JB3 and JB45 [19 (link)] and a fragment of approximately 350 bp of mitochondrial (mt) 12S rDNA was amplified using the primers P60 for and P375 rev [20 (link)]). Primer sequences and PCR details and conditions are given in Additional file 1 : Tables S1 and S2. The amplification products were separated by agarose gel electrophoresis, stained with Midori Green Direct (Nippon Genetics Europe), visualised on a Bio-Rad Gel Doc 1000 (Bio-Rad Laboratories, Hercules, California, USA) and subsequently sent for commercial sequencing in both directions.
5
Subcellular Fractions Proteomics Analysis
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Aliquots (25 μL) of pair-wise subcellular fractions (cytosol and nucleus), corresponding to equal amounts of cells, were mixed with 4 mL glycerol/ 0.1% bromphenol blue (1:1, V/V) and analyzed by SDS-PAGE using a Mini Protean III system (Bio-Rad, Hercules, CA, USA) on a 4 to 15% linear gradient gel. After electroblotting to PVDF membrane (GE-Healthcare, Pollards Woods, UK), proteins were blocked with 5% non-fat dry milk in Tris-buffered saline in the presence of 0.1% Tween (TBS-T) for 1 h at room temperature. Membranes were washed and incubated with primary antibodies overnight at 4°C. Then, membranes were washed with TBS-T and incubated with 1:1,000 dilution of HRP–conjugated secondary antibodies (Sigma, St. Louis, MO, USA) for 1 h at room temperature. After washing with TBS-T, 5-LOX protein was visualized using the HRP substrate ECL Prime (GE-Healthcare, Pollards Woods, UK). Densitometry was performed with a Gel Doc 1000 instrument and the Molecular Analyst software (Bio-Rad, Hercules, CA, USA).
6
Evaluating Immune Signaling Proteins
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The levels of cGAS, STING, HBx, and IRF3 in cells were evaluated by western blotting. Briefly, samples containing an equal amount of protein were separated by SDS-PAGE and blotted onto PVDF membranes. The membranes were blocked with 5% bovine serum albumin and incubated with anti-cGAS, anti-STING, anti-HBx, anti-IRF3, anti-Flag, anti-HA, anti-K48-linked ubiquitin, or anti-β-actin antibodies, followed by incubation with secondary antibodies conjugated with horseradish peroxidase. The proteins of interest were detected using the SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific, USA). The results were recorded by the Bio-Rad Electrophoresis Documentation (Gel Doc 1000, Bio-Rad, USA) and Quantity One Version 4.5.0.
7
GABAC Receptor Plasmid Preparation
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ρ1 GABAC WT and mutant plasmids DNA were linearized by incubation with Xba1. ρ1 WT and mutant cRNA were synthesized using the T7 Transcription mMESSAGE mMACHINE Kit (Ambion, Austin, TX, USA) following the procedure described by the manufacture. The quantity and quality of synthesized CRNA was determined by absorbance at 260–280 nm using Nanodrop Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The purity of synthesized cRNA was confirmed by agarose gel electrophoresis (0.9%) using the GelDoc 1000 (Bio-Rad Laboratories, Hercules, CA USA).
8
Quantitative Analysis of mRNA and miRNA Expression
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Total RNA was isolated from the cells using TRIzol reagent (Ambion, Aukland, New Zealand) according to the manufacturer's instructions for the analysis of mRNA and miRNA expression. The RNA concentration and purity were quantified using a spectrophotometer (NanoDrop 1000 Spectrophotometer; Thermo Fisher Scientific Inc., Waltham, MA, USA). The isolated RNA was used for successive experiments. Total RNA (2,000 ng) was reverse transcribed using reverse transcription primers according to the manufacturer's instructions (Reverse Transcriptase M-MLV, Takara Code: D2639A, Takara Bio Inc., Otsu, Japan). The primers used for RT-PCR and quantitative PCR (qPCR; RT-qPCR) are listed in Table II . The specificity of the PCR products was confirmed by resolving PCR products on 1% agarose gels. The results were recorded using the Bio-Rad Electrophoresis Documentation system (GelDoc 1000; Bio-Rad Laboratories, Hercules, CA, USA) and Quantity One software, version 4.5.0.
9
Epigenetic Regulation of MGMT Gene
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PCR reactions were performed using 2 μl immunoprecipitated DNA, a negative control and a DNA input control. The PCR product length for the MGMT gene fragment was 171 bp and the ChIP-PCR primers, located in the MGMT promoter region, were as follows: Upstream, 5′-CCCCATCTCCAAATAAGGTCA-3′ and downstream, 5′-CCTAGACACTGCCAGAGCCTG-3′. PCR products were resolved on 2% agarose gels (Promega Corporation) and quantified using a GelDoc 1000 (Bio-Rad, Hercules, CA, USA) and Molecular Analyst software (Alpha Innotech Corporation). The levels of H3K9, and H3K4 di-methylation and H3K9 acetylation in each immunoprecipitate were determined by quantifying the intensities of the PCR products in the immunoprecipitated versus input DNA. ChIP was repeated a minimum of two times and three independent PCR analyses of each sample were performed.
10
Protein Extraction and Western Blot Analysis
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Total cellular protein of cervical cancer cells were prepared using RIPA buffer containing phosphatase and protease inhibitors. Nuclear and cytoplasmic proteins were extracted using Nuclear-Cytosol Extraction Kit (cat. no. KGP1100, Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's instructions. Protein extracts were separated by 6-15% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% bovine serum albumin at 37°C for 2 h, washed with Tris-buffered saline with Tween-20 (TBST) and incubated with primary antibodies against S100A9, vimentin, E-cadherin, β-catenin, histone H3, and β-actin separately (1:1,000 dilution) at 4°C overnight. After washing with TBST, the membranes were incubated with the a secondary antibody conjugated with horseradish peroxidase (1:5,000 dilution) for 1 h at 37°C. Finally, the blots were washed with TBST, and then visualized using the Bio-Rad Gel Doc 1000 imaging system and analyzed by Quantity One Version 4.5.0.
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