Whole-cell lysates from frozen tissues and brown adipocytes were isolated using radioimmunoprecipitation assay (RIPA) lysis buffer (150 mmol/L Tris-HCl, 50 mmol/L NaCl, 1% NP-40, 0.1% Tween-20). Protease and phosphatase inhibitors were added to all buffers before experiments. Western blot was performed as previously described [21 (link)]. Protein concentrations were assayed using a Quick StartTM Bradford Assay (Bio-Rad, Hercules, CA, USA). The primary antibodies anti-phosphorylated AMPK (p-AMPK) antibody (Cell Signaling, Danvers, MA, USA), anti-PPARγ antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PGC-1α, and anti-UCP1 antibody (Abcam, Cambridge, UK) were incubated overnight at 4°C, and specific proteins were visualized by the WESTSAVEupTM Detection system (AbFrontier, Seoul, Korea). Band intensities were measured using the GeneTool (SynGene, Cambridge, UK) and normalized to β-actin.
Genetool
Manufactured by Syngene
Sourced in United Kingdom, United States
GeneTool is a versatile and reliable lab equipment designed for molecular biology and genetic research applications. It functions as an automated DNA/RNA extraction and purification system, providing efficient and consistent results. The core function of GeneTool is to isolate and purify nucleic acids from a variety of biological samples, enabling researchers to obtain high-quality genetic material for downstream analysis and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Genesnap, by Syngene (5 mentions)
Pgem t easy vector, by Promega (2 mentions)
T7 and sp6 promoter sequencing primers, by Integrated DNA Technologies (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Anti ucp1 antibody, by Abcam (1 mentions)
Anti pparγ antibody, by Santa Cruz Biotechnology (1 mentions)
Quick starttm bradford assay, by Bio-Rad (1 mentions)
Anti pgc 1α, by Abcam (1 mentions)
Chef dr 3 pulsed field electrophoresis system, by Bio-Rad (1 mentions)
Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)
Chemigenious camera, by Syngene (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Pierce bca protein kit, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (1 mentions)
Gene directory, by Syngene (1 mentions)
Cdna synthesis kit, by Cytiva (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
13 protocols using genetool
1
Protein Expression Analysis in Brown Adipocytes
2
Pulsed Field Electrophoresis for DNA Fragmentation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A CHEF-DR III Pulsed Field Electrophoresis system (Bio-Rad) was used to resolve the DNA. The run time was set to 21 h; temperature was 14°C; the initial and final switch times were 60 and 120 s, respectively; volts/cm was set to 6; the included angle was 120; and 0.5 × TBE was used as the running buffer. The gel was then stained with SYBR Gold Nucleic Acid Gel Stain (Life Technologies) and quantified using Genetool (Syngene) software with the rolling disc method for background subtraction. SYBR Gold Nucleic Acid Gel Stain (Life Technologies) gives a linear relationship between fluorescence intensity and DNA content over at least two orders of magnitude (48 (link)), as also applied previously (49 (link)). The percentage of chromosomal fragmentation was found by first measuring the DNA present in the well and directly beneath the well (non-fragmented DNA and most likely chromosomes with a single nick, respectively) and then measuring the DNA in the rest of the lane. The fragmented DNA value was then divided by the total DNA value. Quantification of the chromosomal fragmentation of a rep recBC mutant with this method was in agreement with already published results (∼50%) (14 (link)).
3
Western Blot Analysis of B-Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
B-cells were harvested and lysed in RIPA-buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5 mM EDTA, 50 mM NaF, 10 mM β-glycerophosphate, 1 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 0.5% aprotinin), and protein concentration was determined by the use of the Pierce BCA protein kit (Thermo Fisher Scientific). Equal amounts of proteins were loaded on and separated by SDS-PAGE. After transfer to an Immobilon–P membrane (Merck Millipore) proteins were detected by standard immunoblotting procedures, using the enhanced chemiluminescence detection system SuperSignal® West Dura Extended Duration substrate (Thermo Fisher Scientific). Images were acquired with a Syngene ChemiGenious camera and presented using GeneSnap (Syngene, Cambridge, England). Signal intensity was calculated using GeneTool (Syngene).
4
3' RACE Protocol for Poly(A) Site Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For 3′RACE, total RNA was reverse transcribed using 3′RACE System for Rapid Amplification of cDNA Ends kits (Life Technologies, USA) with the adapter primer (AP-3′RACE: 5′-AAG CAG TGG TAA CAA CGC AGA GTA CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TVN-3′). Target cDNAs were amplified by PCR and nested PCR with gene-specific forward primers and the amplification primer (AP: 5′-AAG CAG TGG TAA CAA CGC AGA GT-3′). The relative band intensities were measured by densitometry analysis using Gene Tool (Syngene Inc., USA). To identify the Poly(A) sites, bands were excised and extracted using gel extraction kit (Qiagne, USA). Extracted DNAs were cloned into pGEM-T Easy Vector (Promega, USA) according to the manufacturer's protocol. The DNA inserts were sequenced using the customized sequencing primers, T7 and SP6 promoter sequencing primers (Integrated DNA Technologies Inc., USA). The gene-specific forward primers and sequencing primers are as listed in Table S1.
5
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from whole‐cell lysates (15‐30 µg) and mouse tumour samples (50 µg) as previously described.20 , 33 Subcellular fractionation was effected according to supplier instructions (Thermo Fisher, France). Proteins were blotted onto polyvinylidene fluoride membrane (Calbiochem/Merck Millipore) and incubated with primary antibodies. After washing in Tris‐buffered saline/0.1% Tween‐20, immunoreactions were detected as previously described34 using GeneSnap and GeneTool (Syngene, Cambridge, UK). Densitometric analyses were performed using the ImageJ software.
6
Visfatin Promoter Variant Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We extracted nuclear protein from cultured MCF7 cells using the NE-PER nuclear and cytoplasmic extraction kit according to manufacturer’s instructions [ThermoFisher Scientific, USA]. Biotin-labeled probes of the visfatin promoter sequence containing the respective c.-3187G > A and c.-1537C > T variant were synthesized [Integrated DNA technologies, USA]. The probes represent a short section (11 bases) of the visfatin promoter sequence and were designed to include 5 bases before and after the respective SNP. The probe sequences are c.-3187G: 5’-ACTGAG A C T 4 : Figure S4) and the density of each band was quantified by GeneTool [Syngene, Cambridge, England].
7
Estimation of Purified PPO Molecular Weight
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The molecular weight of the partially purified PPO was estimated by SDS–PAGE electrophoresis according to the method of Laemmli [27] (link). The protein was analyzed by SDS–PAGE using 12% polyacrylamide gel, and visualized with Coomassie Blue R-250 staining and its molecular weights were determined by comparing with the molecular weight markers and Genetool, Syngene: (V.4.01).
8
PFGE Analysis of S. aureus Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PFGE patterns were analysed with Gene Tool and Gene Directory software (Syngene). Reference standard S. aureus NCTC 8325 was included in each gel for band normalization. Percent similarities were obtained from the weighted pair group with mathematical average (UPMGA) based on Dice coefficients. Band position tolerance was set at 1.25%. A similarity coefficient of 80% was selected to define the pulsed-field type clusters.
9
Quantitative Analysis of Alternative Splicing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was prepared using Trizol (Invitrogen, USA) according to the manufacturer’s protocol. cDNA was synthesized from 5 μg of total RNA using the cDNA synthesis kit (Amersham Bioscience Inc., USA). cDNAs (150 ng) were used for splicing and qPCR studies with primers and PCR conditions described in Supplementary Table S6 . For the splicing assays, PCR was performed with the indicated primers and the relative band intensities were measured by densitometry analysis using Gene Tool (Syngene Inc., USA). qPCR was performed using the CFX96 Real-time PCR Detection System (Bio-Rad) with the B-R SYBR Green Supermix (Quanta, USA). qPCR was performed to measure the relative inclusion of an alternatively spliced exon by using exon-exon boundary spanning primers that are specific to the alternatively spliced exon and to a constitutive exon. The threshold cycle (Ct) value for each alternatively spliced exon was normalized to the Ct value of the corresponding constitutive exon.
10
Splicing Analysis of Cardiac Transcripts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Heart tissue was pulverized under liquid nitrogen. Total RNA was prepared using Trizol (Invitrogen, USA) according to the manufacturer's protocol. RNA from E18 heart tissues was obtained from Zyagen Inc. cDNA was synthesized from 5 μg of total RNA using the cDNA synthesis kit (Thermo Scientific., USA). cDNAs were used for splicing assays using primers and PCR conditions described in Supplementary Table S6 . The relative band intensities were measured by densitometry analysis using Gene Tool (Syngene Inc., USA). To identify novel splice isoforms, bands were excised and DNA was extracted using gel extraction kits (Qiagen, USA). Extracted DNAs were cloned into pGEM-T Easy Vector (Promega, USA) according to the manufacturer's protocol. The DNA inserts were sequenced using T7 and SP6 promoter sequencing primers (Integrated DNA Technologies Inc, USA). To measure the level of exon 6a inclusion in Scn5a, amplified PCR products were visualized on 2% agarose gel followed by digestion with Sac I (NEB, USA) for 2 h at 37°C. The relative band intensities were measured by densitometry analysis using Gene Tool.
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!