The disruption effects of eltrombopag or DHTS-I were confirmed by EMSA. The reaction system contained 20 nmol/L FAM-labeled ARE and 500 nmol/L HuR protein in the presence or absence of active compounds. The reaction was resolved by a 1% native agarose gel at 4 °C and 50 V for 50 min in 0.5 × TBE buffer as described previously with slight modification25 (link). The resulting gels were visualized by fluorescence imaging using a Typhoon FLA 9500 imager (Typhoon FLA 9500, GE Healthcare, Piscataway, NJ, USA) at wavelength of 470 nm.
Typhoon fla 9500
Manufactured by GE Healthcare
Sourced in United States, Sweden, United Kingdom, Japan, Germany
The Typhoon FLA 9500 is a versatile fluorescence imager designed for a wide range of life science applications. It is capable of capturing high-quality images of fluorescently labeled samples, including gels, blots, and microarrays. The system features a compact design, user-friendly software, and advanced imaging capabilities to support various research and analytical needs.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant software, by GE Healthcare (18 mentions)
Sybr gold, by Thermo Fisher Scientific (13 mentions)
Imagequant, by GE Healthcare (9 mentions)
Imagequant tl, by GE Healthcare (8 mentions)
T4 polynucleotide kinase, by New England Biolabs (7 mentions)
Bis tris gel, by Thermo Fisher Scientific (7 mentions)
Quantity one software, by Bio-Rad (6 mentions)
Storage phosphor screen, by GE Healthcare (6 mentions)
Prism 8, by GraphPad (5 mentions)
Phosphorimager screen, by GE Healthcare (5 mentions)
γ 32p atp, by PerkinElmer (5 mentions)
Imagequant tl software, by GE Healthcare (5 mentions)
Pvdf membrane, by Thermo Fisher Scientific (5 mentions)
Nupage, by Thermo Fisher Scientific (5 mentions)
Imagequant 5, by GE Healthcare (4 mentions)
Mes sds running buffer, by Thermo Fisher Scientific (4 mentions)
Gel doc ez, by Bio-Rad (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
Prism 9, by GraphPad (4 mentions)
Creatine phosphokinase, by Merck Group (3 mentions)
490 protocols using typhoon fla 9500
1
RNA-Protein Binding Assay of HuR
2
Radioactive Kinase Assay Protocol
3
Phosphorylation of TRF1-TERB1 Complex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Co-purified complexes between His6–TRF1TRFH and MBP–TERB1TRFB (9 µM) or MBP–TERB1TBM (130 µM) were incubated with 9 µM phosphorylated CDK1-CyclinB (prepared as previously described58 (link)) in 40 mM Tris pH 7.5, 250 mM KCl, 20 mM MgCl2, 2 mM ATP pH 7.5, 10 mM DTT for 10 min at 30 °C and a further 50 min at 22 °C. Samples were analysed by SEC-MALS and SDS–PAGE with staining by Coomassie and ProQ-Diamond (ThermoFisher), for which imaging was performed using a TyphoonTM FLA 9500 (GE Healthcare), with 532 nm laser, excitation wavelength 555 nm and emission wavelength 580 nm, using the LPG filter and a PMT voltage of 400 V.
4
SNAIL Protein-DNA Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The His6-SNAIL expression vector was constructed using pET28a and transformed into E. coli strain Rosetta (DE3). His6-SNAIL protein expression was induced using 1 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) at 16 °C for 13 h, and the recombinant His-fusion protein was eluted using buffer containing 200 mM imidazole.
The fragment of approximately 30 bp containing putative SNAIL binding sequence in the promoter region (−295 to −260 bp) of FUT8 or its mutant was labeled with FAM at the 5’ end. An EMSA was performed using the Chemiluminescent EMSA Kit (Beyotime, China, Cat# GS005) according to the manufacturer’s protocol. The purified His6-SNAIL fusion protein was incubated with Fam-labeled DNA fragments, and the protein-DNA complexes were separated using 8% PAGE under native conditions, followed by detection on a TyphoonTM FLA 9500 biomolecular imager (GE Healthcare, Piscataway, NJ, USA) using chemiluminescence. A 10-fold molar excess of unlabeled DNA fragments with the same or mutant sequences was used as competitors. The primers used for the EMSA are shown in Supplementary Table4 .
The fragment of approximately 30 bp containing putative SNAIL binding sequence in the promoter region (−295 to −260 bp) of FUT8 or its mutant was labeled with FAM at the 5’ end. An EMSA was performed using the Chemiluminescent EMSA Kit (Beyotime, China, Cat# GS005) according to the manufacturer’s protocol. The purified His6-SNAIL fusion protein was incubated with Fam-labeled DNA fragments, and the protein-DNA complexes were separated using 8% PAGE under native conditions, followed by detection on a TyphoonTM FLA 9500 biomolecular imager (GE Healthcare, Piscataway, NJ, USA) using chemiluminescence. A 10-fold molar excess of unlabeled DNA fragments with the same or mutant sequences was used as competitors. The primers used for the EMSA are shown in Supplementary Table
5
Quantifying Protein-DNA Interactions via EMSA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantification of DNA-binding was performed though EMSA (as described above) using 25 nM FAM-labelled 145 bp random sequence dsDNA, 100 nt poly(dT) or 90 nt random sequence ssDNA at protein concentrations indicated (referring to the molecular oligomeric species). DNA was detected by FAM and SYBR™ Gold (ThermoFisher) staining using a TyphoonTM FLA 9500 (GE Healthcare), with 473 nm laser at excitation wavelength 490 nm and emission wavelength 520 nm, using the LPB filter and a PMT voltage of 400 V. Gels were analysed using ImageJ software (https://imagej.nih.gov/ij/ ). The DNA-bound proportion was plotted against molecular protein concentration and fitted to the Hill equation (below), with apparent KD determined, using Prism8 (GraphPad). Protein concentrations used for apparent KD estimation are quoted for the oligomeric species. DNA sequences are provided in Supplementary Table 3 .
6
In vitro cccDNA formation assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Various RrcDNA substrates (60 ng, 29 fmol, ~2 × 1010 molecules) were incubated with human nuclear extract in reaction buffer containing 20 mM Tris-HCl (pH 7.6), 50 mM KCl, 0.1 mM dNTP, 5 mM MgCl2, 1 mM reduced glutathione, 2.6 mM ATP, 26 mM phosphocreatine disodium (Sigma Aldrich), and 6 μg ml−1 creatine phosphokinase (Sigma Aldrich). The mixture was incubated at 37˚C, and the reaction was terminated by addition of sodium dodecyl sulfate and ethylenediaminetetraacetic acid (EDTA) to final concentrations of 0.5% (w/vol) and 25 mM at indicated time points, respectively. The solution was subsequently treated with proteinase K for 1 hour, and was then extracted by phenol–chloroform and dissolved in RNAse-free water. To monitor cccDNA formation, the repair products were loaded onto a 0.7% (w/vol) agarose gel containing 0.05 μg ml−1 ethidium bromide and run at 4 V/cm for 2 hours before the gel was visualized on TyphoonTM FLA 9500 (GE Healthcare Lifesciences). The intensity of DNA bands was quantified by ImageJ.
7
Quantifying Protein Expression and Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confocal imaging was carried out on living cells as described (see supplementary text)40 (link). Protein amounts were estimated by scanning SDS-PAGEs (10%) with a TyphoonTM FLA9500 gel scanner (GE Healthcare, Sweden) and quantifying YFP- fluorescence with the Fiji gel analysis package. Surface expression of hEAAT1 was quantified with cell surface biotinylation as described previously (see supplementary text)27 (link).
8
Protein-RNA Binding Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein/RNA binding assays were carried out in 1x structure buffer (10 mM Tris-HCl pH 7.0, 100 mM KCl, 10 mM MgCl2) in a volume of 10 μl (30 (link)). Four nM of the α-32P-UTP-labelled heat-denatured RNAs were mixed with 1 μg yeast tRNA (Ambion) and various amounts of the respective proteins (Hfq or Strep-RapZ) and incubated for 30 min at 30°C. The protein dilutions were prepared in 1x structure buffer. Following incubation, 2 μl loading buffer (50% glycerol, 0.5x TBE, 0.2% bromophenol blue) were added and samples were subsequently separated by non-denaturing gel electrophoresis (8% polyacrylamide, 1x TBE) at 300 V for 3 h at 4°C using 0.5x TBE as running buffer. Gels were dried and analysed by phosphorimaging (TyphoonTM FLA 9500, GE Healthcare).
9
Agarose Gel Electrophoresis of PCR Products
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCR products containing 6X DNA loading dye (60 mM EDTA, 10 mM Tris-HCl (pH 7.6), 60 % glycerol, 0.03 % bromphenole blue, 0.03 % xylene cyanol FF, Thermo Scientific) were subjected to horizontal electrophoresis (Owl EasyCastB, Thermo Scientific) and analyzed on either 1.3 % or 2 % agarose gels (containing 0.5x TBE buffer, pH 8). The gels were run at 118 V for ca. 90–120 min.
PCR products were visualized either with GelRed (Biotium, 10 000X in H2O) or by fluorescence imaging of 6-FAM-labeled (6-carboxyfluorescein at 5′-end) oligonucleotides using an electronic dual wave transilluminator equipped with GBox iChemi-XRQ Bio imaging system (Syngene, Life Technologies) or TyphoonTM FLA 9500 (GE Healthcare Life Sciences), respectively.
PCR products were visualized either with GelRed (Biotium, 10 000X in H2O) or by fluorescence imaging of 6-FAM-labeled (6-carboxyfluorescein at 5′-end) oligonucleotides using an electronic dual wave transilluminator equipped with GBox iChemi-XRQ Bio imaging system (Syngene, Life Technologies) or TyphoonTM FLA 9500 (GE Healthcare Life Sciences), respectively.
10
In Vitro Hepatitis B Virus cccDNA Repair
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
60 ng of RrcDNA or NeutrAvidin-RrcDNA complex, or 1–5 ng (estimated by quantitative PCR) of deproteinated virion derived rcDNA were incubated with yeast cell extract in reaction buffer consisting of 20 mM Tris-HCl (pH 7.6), 50 mM KCl, 0.1 mM dNTP, 5 mM MgCl2, 1 mM reduced glutathione, 2.6 mM ATP, 26 mM phosphocreatine disodium (Sigma Aldrich, St. Louis, MO), and 6 μg/ml creatine phosphokinase (Sigma Aldrich, St. Louis, MO). The mixture was incubated at 30°C for 60 min before the reaction was stopped by addition of SDS and EDTA to final concentrations of 0.5% (w/vol) and 25 mM, respectively. The solution was subsequently treated with RNase A and proteinase K for 1 hour each. The solution was then extracted by phenol-chloroform (1:1, vol/vol) and repair product precipitated by ethanol and dissolved in TE buffer consisting of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. To monitor cccDNA formation, the repair products were loaded onto a 0.7% (w/vol) agarose gel containing 0.05 μg/ml ethidium bromide and run at 4 V/cm for 2 hours before the gel was visualized on Typhoon™ FLA 9500 (GE Healthcare Lifesciences, Chicago, IL). The intensity of DNA bands was quantified by ImageJ.
For the repair reaction using deproteinated rcDNA (dp-rcDNA), cccDNA was detected by Southern blotting as describe in the Southern blotting section.
For the repair reaction using deproteinated rcDNA (dp-rcDNA), cccDNA was detected by Southern blotting as describe in the Southern blotting section.
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!