Whole-cell lysates were rapidly prepared by adding lysis buffer (4% SDS and 720 mM 2-mercaptoethanol, preheated to 100 °C) directly to cell monolayer; the lysate was recovered and heated for 5 more minutes after the addition of glycerol to 10%. For instances in which cell adhesion was compromised (e.g., downregulation of SRSF1 in SUM159PT cells and treatment with nocodazole in T47D cells), care was taken (using centrifugation) to ensure recovery of loosely adherent or floating cells. Equivalent aliquots (by protein content, unless otherwise indicated) were separated on 10% SDS/PAGE gels, transferred to 0.2-μm nitrocellulose (BioRad, cat# 1620146) or PVDF (BioRad, cat#1620174) membranes, and subjected to standard immunoblotting procedures followed by chemiluminescence (GE Healthcare, cat# RPN2109) image capture. Image analysis and band densitometry was performed using the Genetools Analysis software (Syngene, Frederick, MD).
Genetools analysis software
Manufactured by Syngene
Sourced in United Kingdom, United States
GeneTools analysis software is a computer program designed to analyze and interpret data from various molecular biology experiments, such as DNA sequencing, gene expression, and protein analysis. The software provides a range of tools for data visualization, statistical analysis, and bioinformatics.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (12 mentions)
Hiseq 2500 platform, by Illumina (10 mentions)
Nebnext ultra dna library prep kit for, by Illumina (8 mentions)
Nanodrop one, by Thermo Fisher Scientific (8 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (4 mentions)
Premix taq, by Takara Bio (4 mentions)
Miseq pe300, by Illumina (3 mentions)
S1000, by Bio-Rad (2 mentions)
Nebnext ultra 2 dna library prep kit for, by Illumina (2 mentions)
E z n a gel extraction kit, by Omega Bio-Tek (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Nucleic acid stain 1, by Roche (2 mentions)
Nebnext ultra dna library prep kit, by Illumina (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (2 mentions)
12 bp barcoded primers, by Thermo Fisher Scientific (2 mentions)
Magabio soil feces genomic dna purification kit, by Bioer (2 mentions)
Sybr safe nucleic acid stain, by Thermo Fisher Scientific (2 mentions)
G box chemi imager, by Syngene (2 mentions)
Nanodrop one spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
54 protocols using genetools analysis software
1
Rapid Whole-Cell Lysis for Immunoblotting
2
Western Blot Analysis of Alzheimer's Proteins
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Samples were made up in dissociation buffer (1× dissociation buffer (100 mm Tris-HCl, 2% (w/v) SDS, 10% (v/v) glycerol, 100 mm DTT, 0.02% (w/v) bromphenol blue, pH 6.8) and heated at 95 °C for 5 min. Proteins were resolved by SDS-PAGE on 7–17% acrylamide Tris-glycine gels and then transferred to Hybond polyvinylidene difluoride membranes (GE Healthcare). Following electrotransfer, the membranes were blocked for 1 h in PBS with 0.1% Tween 20 (PBST) and 5% (w/v) nonfat milk and then incubated with primary antibody overnight at 4 °C. Antigens were probed using the following primary antibodies: anti-APP (22C11, Millipore), SAF32 (anti-PrP N terminus, Cayman Chemical), 8H4 (anti-PrP residues 175–185), anti-ADAM10 antibody (Abcam), 6E10 (anti-Aβ(1–17), Merck Biosciences), AC15 (anti-β-actin) and synapsin 1 (Sigma), 2B3 (anti-human sAPPα, Immuno-Biological Laboratories), and anti-phospho-Src family kinase (Tyr-416; Cell Signaling Technology). Primary antibodies were detected by incubation with horseradish peroxidase–conjugated secondary antibody, both in PBST containing 2% BSA. Bound horseradish peroxidase conjugates were visualized using the ECL® detection system with a Syngene Gbox XT4 (Syngene). Densitometric analysis was performed using Genetools analysis software (Syngene).
3
Gut Microbiome Profiling Using 16S rRNA Sequencing
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The microbial DNA (with a total mass of 1.2–10.0 ng) was isolated from each fecal sample using the MOBIO Pow erSoil DNA Isolation Kit and was quantified with NanoDrop One (Thermo Fisher Scientific, Waltham, MA, United States). The V4 regions of the DNA genes were amplified by using the specific primer 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTW TCTAAT-3′) with 12 bp barcode. Primers were synthesized by Invitrogen (Invitrogen, Carlsbad, CA, United States). The PCR instrument was Bio-Rad S1000 (Bio-Rad Laboratory, CA, United States). The length and concentration of the PCR product were detected by 1% agarose gel electrophoresis. PCR products with bright main strip between were mixed in equidensity ratios according to the GeneTools Analysis Software (Version 4.03.05.0, SynGene). Then, mixture of PCR products was purified with E.Z.N.A. Gel Extraction Kit (Omega, United States). Sequencing libraries were generated using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, MA, United States) following the manufacturer’s recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific, MA, United States). At last, the library was sequenced on an Illumina Nova6000 platform and 250 bp paired-end reads were generated (Guangdong Magigene Biotechnology Co., Ltd. Guangzhou, China).
4
Protein Expression Analysis by Western Blot
5
FKBP52 Protein Expression Analysis
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For each case studied (Table 2 ), a portion of medial frontal cortex (F2) was homogenized at 4°C using a Dounce homogenizer (glass-Teflon) in 5 volume (w/v) of buffer consisting of: Tris 10 mM, saccharose 0.32 M, and DTT 1 mM at pH 7.4 with “Complete” protease inhibitor cocktails (Roche). Homogenates were centrifuged 5 min at 1000 g and the protein estimation of the upper fraction was performed using a BCA protein assay kit (Pierce). Samples were analyzed on a 10% SDS Page gel and transferred onto iBlot™ Gel Transfer membranes (Invitrogen). Following transfer, membranes were incubated overnight with appropriate antibodies [FKBP52, mouse monoclonal EC1 (1 : 1000) (Enzo Life Sciences) and a Rabbit polyclonal HRP conjugated GAPDH (1 : 1000) (Abcam) rather than Actin for the loading control as recommended [41 (link)]. Images were recorded with the GeneGnome5 (Syngene). Quantification was performed using Genetools analysis software (Syngene).
6
Amplicon Sequencing of Microbial 16S rRNA
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DNA was extracted using DNA extraction kit (Minkagene Stool DNA kit) for the corresponding sample. The concentration and purity were measured using the NanoDrop One (Thermo Fisher Scientific, MA, USA). 16S rRNA genes of distinct region (V4) were amplified using specific primers (515F and 806R) with 12 bp barcodes. Primers were synthesized by Invitrogen (Carlsbad, CA, USA). PCR reactions, containing 25 μl 2 × Premix Taq (Takara Biotechnology, Dalian Co. Ltd., China), the PCR instrument was BioRad S1000 (Bio-Rad Laboratory, CA). The length and concentration of the PCR product were detected by 1% agarose gel electrophoresis. PCR products were mixed in equimolar ratios according to the GeneTools Analysis Software (Version4.03.05.0, SynGene). Then, the PCR mixture was purified with EZNA Gel Extraction Kit (Omega, USA). Then, sequencing libraries were generated using NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs, USA) following the manufacturer’s recommendations and index codes were added. The library quality was assessed on the Qubit 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Lastly, the library was sequenced on an Illumina Hiseq 2500 platform and 250 bp paired-end reads were generated.
7
16S rRNA Amplification and Illumina Sequencing
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The hypervariable V4-V5 regions of the 16S rRNA genes from bacteria and archaea were amplified in triplicate by PCR using the barcoded primers 515-F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 907-R (5′-CCGTCAATTCMTTTRAGTTT-3′) (Invitrogen, Thermo Fisher Scientific Inc., Fair Lawn, USA). PCR was performed on a thermocycler using Premix Taq (EX Taq Version 2.0 plus dye, Takara Biotechnology Co. Ltd., Dalian, China) with following conditions: 94 °C for 5 min followed by 30 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 30 s and a final extension at 72 °C for 10mins. The PCR products were verified by 1% agarose gel electrophoresis and quantified using GeneTools analysis software (Version 4.03.05.0, SynGene, Frederick, USA). Technical triplicate amplicons were pooled and gel purified using EZNA Gel Extraction Kit (Omega Bio-tek, Inc., Norcoross, USA). Sequencing was performed using 250-bp paired-end strategy on an Illumina HiSeq 2500 platform (Illumina Inc., San Diego, USA) with NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, USA).
8
Determining IC50 and Protein Expression
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Non-linear regression graph plots for determining the IC50 and statistical analyses were performed using GraphPad Software Inc. (San Diego, CA; www.graphpad.com ) Prism version 5.00 for Windows, was used.
For gel electrophoresis applications, inGenius—gel documentation system comprising of GeneTools analysis software (Syngene, MD, USA) was used for digital imaging and relative sample expression levels.
For gel electrophoresis applications, inGenius—gel documentation system comprising of GeneTools analysis software (Syngene, MD, USA) was used for digital imaging and relative sample expression levels.
9
Western Blot Analysis of Cyclin D1 Isoforms in Thyroid Cancer
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Human thyroid cancer cell lines were homogenized in radio-immunoprecipitation assay buffer (Thermo Scientific, Fremont, CA, USA) and a mixture of 5 mM EDTA and protease inhibitors (Thermo Scientific). Protein extracts were separated on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany). After blocked in Starting Block T20 (TBS) Blocking buffer (Thermo Scientific), the membrane was incubated with primary antibodies (cyclin D1a, 1:1500; cyclin D1b, 1:1000) for 16 h at 4 °C on an orbital shaker. Primary antibodies were diluted in the blocking buffer. The membrane was washed three times for 10 min with Tris-buffered saline with Tween 20 (TBST, Thermo Scientific), followed by incubation with goat anti-rabbit IgG horseradish peroxidase conjugated antibody (1:2500, Santa Cruz Biotechnology, CA, USA) for 60 min at room temperature (22–25 °C) on an orbital shaker. The detection of immunoreaction was achieved using an enhanced SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Scientific). Immuno-reactive bands were visualized using the PXi Touch Imaging System (Syngene, Frederick, MD, USA) and quantified using the GeneTools Analysis Software (Syngene).
10
Amplification and Sequencing of 16S rRNA Genes
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The hypervariable V4–V5 regions of the 16S rRNA genes from bacteria and archaea were PCR amplified in triplicate using the barcoded primers 515-F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 907-R (5′-CCGTCAATTCMTTTRAGTTT-3′). PCR amplifications were performed with the following thermocycling conditions: 94 °C for 5 min followed by 30 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 30 s and a final extension at 72 °C for 10 min. The PCR products were verified by 1% agarose gel electrophoresis and quantified using GeneTools Analysis Software (Version 4.03.05.0, SynGene, Frederick, MD, USA). Technical triplicate amplicons were pooled and gel purified using the EZNA Gel Extraction Kit (Omega Bio-tek, Inc., Norcoross, GA, USA). A gene library was constructed using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA, USA). Sequencing was performed on an Illumina HiSeq 2500 platform (Illumina, Inc., San Diego, CA, USA) according to the manufacturer’s instructions and 250 bp paired-end reads were generated.
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