GB cells were washed with PBS and then lysed in RIPA lysis buffer. Cellular protein lysates were isolated using Protein Extraction Kit (Qiagen, Germantown, MD, USA) and quantified using Bradford Protein Assay Kit (Qiagen). In total, 20 μg of samples from different experiments were loaded and subjected to SDS-PAGE using the Mini-Protean III system (Bio-Rad, Taipei City, Taiwan). Separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes using Trans-Blot Turbo Transfer System (Bio-Rad) followed by blocking with Tris-buffered saline plus skim milk. Then, these PVDF membranes were probed with respective primary antibodies followed by a secondary antibody. The primary antibodies for CD133, KLF4, and SOX2 are shown in Supplementary Table S1 . ECL detection kit was used for detecting proteins of interest. Images were captured and analyzed using an UVP BioDoc-It system (Analytik Jena, Thuringia, Germany). RT-qPCR was performed using isolated total RNA according to the TRIzol-based protocol (Life Technologies, Carlsbad, CA, USA) provided by the manufacturer. One microgram of total RNA was reverse transcribed using a Qiagen OneStep RT-PCR Kit (Qiagen), and the PCR reaction was performed using a Rotor-Gene SYBR Green PCR Kit (400, Qiagen).
Biodoc it system
Manufactured by Analytik Jena
Sourced in United States
The BioDoc-It System is a versatile lab equipment designed for imaging and documentation of nucleic acid and protein gels, blots, and other samples. It features a high-resolution camera and illumination options to capture high-quality images for analysis and archiving.
Automatically generated - may contain errors
Lab products found in correlation
Mini protean 3 system, by Bio-Rad (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Protein extraction kit, by Qiagen (1 mentions)
Rotor gene sybr green pcr kit, by Qiagen (1 mentions)
Bradford protein assay kit, by Qiagen (1 mentions)
Trizol based protocol, by Thermo Fisher Scientific (1 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
Blocking agent, by GE Healthcare (1 mentions)
Fla 2000, by Fujifilm (1 mentions)
Trans blot semi dry electrophoretic transfer cell, by Bio-Rad (1 mentions)
Hybond n, by GE Healthcare (1 mentions)
Sybr gold, by Thermo Fisher Scientific (1 mentions)
7 protocols using biodoc it system
1
Protein Expression Analysis of Stem Cell Markers
2
ChIP Assay of TBX1 Transcription Factor
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The ChIP assay was performed as previously described48 (link), with minor modifications. After cells were formaldehyde cross-linked and lysed, sheared chromatin was immunoprecipitated with anti-TBX1 antibody (34–9800, ThermoFisher), and DNA was isolated and analyzed by PCR. The gels were imaged using BioDoc-It System (analytik jena US). The primer sequences are listed in Supplementary data, Table S3 . The full-length gel is shown in Supplementary data, Fig. S14 .
3
RNA to cDNA Conversion and qPCR
4
DNA Isolation and Characterization from Liver
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The DNA was isolated from the liver of the different groups by ZR Genomic DNATM-Tissue MidiPrep, Irvine, CA 92614, USA, according to manufacturer's instructions. The quality and quantity of DNA was evaluated spectrophotometrically at 260/280 nm. DNA samples were analyzed on a standard 1% (w/v) agarose gel containing ethidium bromide. Images of the ethidium bromide stained DNA agarose gel were acquired using UVP BIODOC-IT™ system.
5
Protein Expression Analysis of BC-N102-Treated MCF-7 Cells
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Cell lysates from BC-N102-treated MCF-7 cell lines, were subjected to protein expression analyses using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a Mini-Protean III system (Bio-Rad, Taipei, Taiwan) and transferred onto polyvinylidene difluoride membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were incubated with primary antibodies for the PI3K (CAT: #E11-1224B, 1:1000), p-ERK (CAT: #4370, 1:1000), p-AKT (CAT: #4060, 1:1000), P38 MAPK (1:1000), CDK2 (CAT: #2546, 1:1000), CDK4 (CAT: #2906, 1:1000), cyclin D1 (CAT: #2926, 1:1000), p-CDC35C (1:1000), p21 (CAT: #2947, 1:1000), p27 (CAT: #2552, 1:1000), p53 (CAT:2557 #1:1000), and GAPDH (CAT: #10494-1-AP 1:1000) for 24 h at 4 °C, followed by incubation with the respective secondary antibodies at room temperature for 1 h. Protein signals were detected and visualized with the aid of an enhanced chemiluminescence (ECL) detection kit, and images were captured using the UVP BioDoc-It system (Upland, CA, USA).
6
Native PAGE Separation and Detection of RNA:DNA Hybrids
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Size separation of nucleic acids under native conditions was performed using 15% native PAGE gels, pre-run for 1 h at 5 W 4°C, samples were loaded in an equal volume of native loading buffer (30% (v/v) glycerol, 80 mM HEPES-KOH (pH 7.9), 100 mM KCl, 2 mM magnesium acetate) and electrophoresed in 1× TBE at 5 W 4°C in a vertical gel tank (EV200, Cambridge Electrophoresis). Nucleic acids were visualised using Sybr Gold (Invitrogen) according to manufacturer's instructions and imaged on a phosphorimager (FLA-2000, Fujifilm) or UV transilluminator (BioDoc-It System, UVP). Immunoblot detection of RNA:DNA hybrids was performed by transfer of electrophoresed nucleic acids to Hybond-N+ (GE Healthcare) using a Trans-Blot® Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories), in 1× TBE for 30 min at a maximum current of 3 mA/cm2. Nucleic acids were cross-linked to the membrane by exposure to 1200 mJ/cm2 UV (Stratalinker, Stratagene). Membrane was pre-blocked overnight in 1% (w/v) blocking agent (GE Healthcare) in 0.1% (v/v) Tween-20/PBS at 4°C, washed in 0.1% (v/v) Tween-20/PBS, probed with 200 ng/ml S9.6 antibody in 2% (w/v) BSA/PBS overnight at 4°C, and then incubated with HRP-conjugated goat anti-mouse IgG antibody (1:5,000, Dako Corporation) for 1 h at room temperature, followed by ECL detection (GE Healthcare).
7
Genotyping by Restriction Fragment Length Polymorphism
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The amplified 331 bp PCR product (3 µl) was digested in a 10-µl final reaction volume using 1 µl of Reaction Buffer 2 and 5 units of MvaI restriction enzyme (New England Biolabs, Beverly, MA, USA) at 37°C overnight. Controls of known genotype were included for every set of digestions carried out. After digestion, allele G yielded three fragments of 99, 188, and 44 bp, whereas allele A yielded two fragments of 99 and 232 bp. Digested fragments were separated on 3% agarose gels and visualized after ethidium bromide staining in the BioDoc-It System (UVP, Upland, CA, USA) Bioimaging system.
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