1-Step Human Coupled IVT Kit – DNA (Thermo Scientific) was used to carry out experiments. 0.5 μg of pCFE-GFP plasmid was used for all reactions and compounds were used at 5 μM. Compounds were irradiated with one minute indigo light (29.1 J/cm2) then incubated with plasmid overnight. The reaction mix was made according to the procedure described by Thermo Scientific and all reactions were scaled down to 12.5 μL. Reactions were incubated in a water bath for two hours at 30°C then read in a Greiner-Bio One 384-well small volume plate. All samples were read on a SpectraFluor Plus (Tecan) Plate Reader with a 485 nm excitation and 530 nm emission.
1 step human coupled ivt kit dna
Manufactured by Thermo Fisher Scientific
The 1-Step Human Coupled IVT Kit – DNA is a laboratory product designed for in vitro transcription and translation of DNA templates. It provides a streamlined process for generating human proteins from DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Kinase buffer, by Cell Signaling Technology (3 mentions)
Pt7cfe1 chis plasmid, by Thermo Fisher Scientific (3 mentions)
His pur cobalt spin columns, by Thermo Fisher Scientific (3 mentions)
Adp glo kinase assay kit, by Promega (2 mentions)
Prism 9, by GraphPad (2 mentions)
Spectrafluor plus, by Tecan (1 mentions)
Energy regeneration solution, by R&D Systems (1 mentions)
Amp glo assay, by Promega (1 mentions)
Infinite f200 pro, by Tecan (1 mentions)
Adenosine triphosphate (atp), by Sino Biological (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
The his tag antibody, by GenScript (1 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
10 protocols using 1 step human coupled ivt kit dna
1
Optimized GFP Plasmid Photoreaction
2
In vitro Ubiquitination Assay for UBE2A
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro translation of UBE2A proteins was performed with 1-Step Human Coupled IVT Kit-DNA (Thermo-Fischer-Scientific) following the manufacturer’s instructions. For ubiquitination assay we incubated 15 μg of UBE2A proteins with 1 μg of GST-Ubiquitin (Enzo-Life-Sciences, NY, USA), 0.2 ng of ubiquitin activating enzyme (E1) (Enzo-Life-Sciences), 2 mM ATP, energy regeneration solution (BostonBiochem, MA, USA), 2 mM MgCl2, 2 mM KCl, 16 μg of BA/F3_BCR-ABL1 whole cell lysate in 50 mM TrisHCl (ph7.5). The reactions were incubated for 20 minutes at 37°C. The products were analyzed by western blotting.
For the enzymatic activity of WT and mutated UBE2A, 15 μg of UBE2A in vitro synthesized protein were used. The AMP-Glo Assay (Promega catalog v5011) was used in order to quantify the amount of AMP generated by the ubiquitin conjugation machinery, composed of 170 ng/μL ubiquitin protein, 15 ng/μL UBA1 and 50 μM ATP (SignalChem).
The production of AMP from ATP is directly proportional to the enzymatic activity of the ubiquitination machinery and therefore it was used to measure the ubiquitination in the presence of WT and mutated UBE2A. The AMP signal was detected using the AMP detection solution (Promega) and a TECAN reading plate (Infinite F200Pro TECAN).
For the enzymatic activity of WT and mutated UBE2A, 15 μg of UBE2A in vitro synthesized protein were used. The AMP-Glo Assay (Promega catalog v5011) was used in order to quantify the amount of AMP generated by the ubiquitin conjugation machinery, composed of 170 ng/μL ubiquitin protein, 15 ng/μL UBA1 and 50 μM ATP (SignalChem).
The production of AMP from ATP is directly proportional to the enzymatic activity of the ubiquitination machinery and therefore it was used to measure the ubiquitination in the presence of WT and mutated UBE2A. The AMP signal was detected using the AMP detection solution (Promega) and a TECAN reading plate (Infinite F200Pro TECAN).
3
In Vitro Synthesis and Detection of LOC646736 Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 1-step Human Coupled IVT Kit-DNA (Thermo Scientific) was used for in vitro synthesis of LOC646736 proteins [19 (link)]. Circular plasmids (1 μg) pCFE-LOC646736_ex1-3 and pCFE-LOC646736_ex5-7 were each incubated with 12.5 μL HeLa Lysate, 2.5 μL accessory proteins, and 5 μL reaction mix in a total volume of 25 μL for 90 min at 30°C. Samples were denatured in sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM dithiothreitol, and 0.01% bromophenol blue) at 70°C for 10 min, cooled on ice, and subjected to electrophoresis in 12% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Life Sciences, Buckinghamshire, England). Membranes were blocked with 5% nonfat dry milk diluted in wash buffer (0.1% Tween-20 in Tris-buffered saline, pH 7.6) for 2 h at room temperature, incubated with THE His Tag Antibody (GenScript, Piscataway, NJ, USA) overnight at 4°C, washed 5 times, and incubated with the secondary antibody, IRDye 800CW goat anti-mouse IgG (Li-cor, Superior St. Lincoln, NE, USA) for 1 h at room temperature. After three 5-min incubations with wash buffer, proteins were visualized using the Odyssey infrared imaging system (Li-COR).
4
In vitro Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro coupled transcription and translation analysis was performed with the 1-Step Human Coupled IVT Kit ± DNA (ThermoFisher Scientific) following the manufacturer’s instructions. Briefly, the translation reaction was assembled with pre-incubation of HeLa cell lysates with 1 μg of circular DNA (pCFE-GFP) as a template. The reaction mixtures were incubated for 12 hours at 30 °C and were stopped with loading buffer for SDS-PAGE. The expression levels of EGFP proteins were examined by immunoblot analysis using anti-TurboGFP antibody. For in vitro translational analysis, EGFP mRNA was synthesized after digesting pcGlobin2-EGFP with XhoI, as described previously60 (link). Synthetic EGFP mRNA was generated by using a mMESSAGE mMACHINE T7 Transcription kit (ThermoFisher Scientific) following manufacturer’s instructions. Each 3 µg of purified EGFP mRNA was incubated with HeLa cell lysates for 6 hours at 30 °C with indicated concentrations of PINO and CHX as described in figure legends. The expression level of EGFP was measured through immunoblotting with anti-GFP antibody and then quantified by densitometry using an image J software.
5
In vitro Tau Translation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The in vitro translation assay was done using 1-Step Human Coupled IVT Kit – DNA; 88881, ThermoFisher Scientific. K18 and Ht40 were added to the prescribed reaction mix to a final concentration of 50 µM. The positive control set contained no tau proteins and the negative control set did not contain the GFP (reporter gene) plasmid. All the reaction sets were incubated till 6 hours at 30 °C (as prescribed) and GFP (reporter gene) fluorescence was monitored at ex/em: 482/512 nm. The experiment was repeated three times.
6
In vitro Expression of PU.1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
In vitro transcription/translation (IVT) samples of PU.1 (full-length, untagged) were generated using 1-Step Human Coupled IVT Kit – DNA (Thermo Fisher Scientific Cat# 88881) following the provider's instructions. Protein expression was confirmed by western analysis.
+ Expand
In vitro transcription/translation (IVT) samples of PU.1 (full-length, untagged) were generated using 1-Step Human Coupled IVT Kit – DNA (Thermo Fisher Scientific Cat# 88881) following the provider's instructions. Protein expression was confirmed by western analysis.
7
In vitro Protein Synthesis from Plasmids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro coupled transcription and translation of plasmids was performed with the 1-Step Human Coupled IVT Kit–DNA (Thermo Scientific) following the manufacturer’s instructions. 1 μg of circular DNA or 1 μg of in vitro synthesized mRNAs were used as templates. The reaction mixtures were incubated for 6 h at 30°C and were stopped with 8 μl loading buffer for PAGE and Western Blot analysis.
8
Purification and Phosphorylation of Recombinant OCT2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Yes1 recombinant human protein was obtained from Life technologies (A15557). To purify OCT2 proteins, FLAG-tagged wild-type or mutant OCT2 constructs (described in the site-directed mutagenesis section) were subcloned into pT7CFE1-CHis plasmid (Thermo Fischer). These constructs were then used for in vitro translation using a HeLa cell lysate-based Kit (1-Step Human Coupled IVT Kit – DNA; 88881, Life Technologies). The in vitro translated proteins were then purified using His Pur cobalt spin columns (Thermo Scientific). For in vitro kinase assays, recombinant Yes1 and purified OCT2 proteins were incubated in a kinase buffer (Cell Signaling, 9802) supplemented with cold ATP (Cell signaling, 9804) at 30 °C for 30 min. After the incubation period, the reaction was terminated and OCT2 proteins were immunoprecipitated by FLAG tagged beads (described in the Protein Analysis section) followed by western blot analysis to determine OCT2 tyrosine phosphorylation.
9
In Vitro CDKL5 Kinase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Methods used for these assays have been described.28 (link) Briefly, human FLAG-tagged CDKL5 WT or kinase dead (KD, CDKL5 K42R) constructs were subcloned into pT7CFE1-CHis plasmid (Thermo Fisher). These constructs were then used for in vitro translation using a HeLa cell lysate-based Kit (1-Step Human Coupled IVT Kit—DNA, 88881, Life Technologies). The in vitro-translated proteins were then purified using His Pur cobalt spin columns (Thermo Scientific). For in vitro kinase assays, recombinant CDKL5 and myelin basic protein (Active Motif, 31314) as a substrate were incubated in a kinase buffer (Cell Signaling, 9802) supplemented with or without 50 µM adenosine 5′-triphosphate (ATP) at 30 °C for 30 minutes followed by kinase assays using ADP-Glo Kinase Assay kit (Promega). Data were analyzed using GraphPad Prism 9. Each assay was run in triplicate (n = 3) and mean values are graphed in Figure 7 . All error bars in Figure 7 are standard deviation (SD). Statistical analysis was done using one-way ANOVA with Dunnett’s multiple comparisons test. Statistical methods and p-values are mentioned in the Figure 7 legend. Thresholds for significance were placed at ***p <0.0001. Non-significant statistics were not indicated.
10
In Vitro Kinase Assay for CDKL5
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Methods used for these assays
have been described.30 (link) Briefly, human FLAG-tagged
CDKL5 WT or KD (CDKL5 K42R) constructs were subcloned into pT7CFE1-CHis
plasmid (Thermo Fisher). These constructs were then used for in vitro
translation using a HeLa cell lysate-based kit (1-Step Human Coupled
IVT Kit—DNA, 88881, Life Technologies). The in vitro-translated
proteins were then purified using HisPur cobalt spin columns (Thermo
Scientific). For in vitro kinase assays, recombinant CDKL5 and myelin
basic protein (Active Motif, 31314) as a substrate were incubated
in a kinase buffer (Cell Signaling, 9802) supplemented with or without
50 μM adenosine 5′-triphosphate (ATP) at 30 °C for
30 min followed by kinase assays using the ADP-Glo kinase assay
kit (Promega). Data were analyzed using GraphPad Prism 9. Each assay
was run in triplicate (n = 3), and the mean values
are graphed inFigure 7 . All error bars in Figure 7 are standard deviation (SD). Statistical analysis was done
using one-way ANOVA with Dunnett’s multiple comparisons test.
Statistical methods and p-values are mentioned in
theFigure 7 legend.
Thresholds for significance were placed at ***p <
0.0001. Non-significant statistics were not indicated.
have been described.30 (link) Briefly, human FLAG-tagged
CDKL5 WT or KD (CDKL5 K42R) constructs were subcloned into pT7CFE1-CHis
plasmid (Thermo Fisher). These constructs were then used for in vitro
translation using a HeLa cell lysate-based kit (1-Step Human Coupled
IVT Kit—DNA, 88881, Life Technologies). The in vitro-translated
proteins were then purified using HisPur cobalt spin columns (Thermo
Scientific). For in vitro kinase assays, recombinant CDKL5 and myelin
basic protein (Active Motif, 31314) as a substrate were incubated
in a kinase buffer (Cell Signaling, 9802) supplemented with or without
50 μM adenosine 5′-triphosphate (ATP) at 30 °C for
30 min followed by kinase assays using the ADP-Glo kinase assay
kit (Promega). Data were analyzed using GraphPad Prism 9. Each assay
was run in triplicate (n = 3), and the mean values
are graphed in
using one-way ANOVA with Dunnett’s multiple comparisons test.
Statistical methods and p-values are mentioned in
the
Thresholds for significance were placed at ***p <
0.0001. Non-significant statistics were not indicated.
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!