To obtain purified YL2-His protein, full-length YL2 cDNA was amplified using the primers YL2-HIS-F and YL2-HIS-R (Supplementary Table 2) then subcloned into the vector pET44a. The resulting construct and empty vector were expressed in Escherichia coli strain BL21. Recombinant YL2-His was purified using Ni-NTA resin (GenScript) and used for kinase assay. The UMP kinase assay was performed as described previously (Yoshida et al. 2012 (link)). The 100 μL assay mixture consisted of 50 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 0.2 mM UMP, and 10 μM ATP. The reaction was started by the addition of various amounts of purified His-tagged YL2 protein and incubated at 30 °C for 0.5 h. Next, 100 μL of the Kinase-Glo Assay reagent was added to initiate the luciferase reaction. The luminescence intensity was measured after 10 min incubation at room temperature using an ARVO luminometer (PerkinElmer, America).
Arvo luminometer
Manufactured by PerkinElmer
Sourced in United States
The ARVO luminometer is a versatile instrument designed for measuring luminescence signals. It is capable of detecting and quantifying various luminescent reactions, including bioluminescence, chemiluminescence, and fluorescence. The ARVO luminometer offers high sensitivity and a wide dynamic range, making it suitable for a range of applications in life science research and diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Fugene 6, by Promega (2 mentions)
Nepa21 super electroporator, by Nepa Gene (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Ni nta resin, by GenScript (1 mentions)
Ofcelltiter glo reagent, by Promega (1 mentions)
Nano glo luciferase assay system, by Promega (1 mentions)
Bz x700 microscope, by Keyence (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Nepa21, by Nepa Gene (1 mentions)
Nano glo dual luciferase reporter assay system, by Promega (1 mentions)
Qubit dsdna hs quantitation assay, by Thermo Fisher Scientific (1 mentions)
Recombinant amv rtα, by Nippon Gene (1 mentions)
Complete mini edta free protease inhibitor cocktail, by Merck Group (1 mentions)
Ur 21p, by TOMY (1 mentions)
6 protocols using arvo luminometer
1
Purification and Kinase Assay of YL2-His
2
Cytotoxicity Assay for POM Esters
3
Stable Transfection and Selection of Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SaPe-4, S2 and Sf9 cells were transfected with the vectors containing AcGFP1- and ZeoR-expression cassettes using FuGENE 6 (Promega). Five days after transfection, the SaPe-4 and Sf9 cells were treated with 100 µg/ml zeocin, while S2 cells were treated with 400 µg/ml zeocin. After three weeks of selection in zeocin-containing medium, the cells were subjected to flow cytometry analysis and image acquisition by a CytoFLEX S (Beckman Coulter Life Sciences, Indianapolis, IN, USA) and a BZ-X700 microscope (Keyence, Osaka, Japan), respectively. Similarly, stable Nluc-expressing S2 cells were established. Nluc activity was measured using an ARVO luminometer (PerkinElmer) with the Nano-Glo® Luciferase assay system (Promega).
4
Transfection of Insect Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfection into Pv11 cells was carried out using a NEPA21 Super Electroporator (Nepa Gene, Ichikawa, Chiba, Japan) as described previously7 (link). S2, SaPe-4, AeAl-2, Sf9 and BmN4 cells were seeded at a density of 5.0 × 105 cells per well in 6-well plates. The next day, 0.75 µg MetLuc-reporter vector and 0.75 µg SEAP-reference vector were transfected with 1.25 µl FuGENE6 (Promega). The medium was collected 72 h later, and luciferase activity was measured using an ARVO luminometer (PerkinElmer, Waltham, MA, USA) with the Ready-To-Glow dual secreted reporter assay system (TaKaRa Bio). For Tc81 transfection, 10 µg MetLuc-reporter vector and 10 µg SEAP-reference vector were transfected into 2.5 × 105 cells using NEPA21 (Nepa Gene). The electroporation conditions were as follows: poration pulse (pulse voltage, 175 V; pulse width, 4 ms; pulse interval, 50 ms; pulse number, 6; voltage decay, 10%; voltage polarity, +) and transfer pulses (pulse voltage, 20 V; pulse width, 50 ms; pulse interval, 50 ms; pulse number, 5 for each polarity; voltage decay, 40%; voltage polarity, +/−). To assess transfection efficiency, SEAP reference vectors were used. The promoter for the reference vector in each experiment was as follows: PvGapdh promoter in Fig. 1a, c, d ; Bmhsp90 promoter in Figs. 1b and 2 ; OpIE2 promoter in Fig. 3 .
5
Luciferase Assay Protocol for Pv11 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells used in each experiment were seeded at a density of 3 × 10 5 cells per mL into a 25 cm 2 cell culture flask and grown at 25℃ for 6 days before transfection. Transfection into Pv11 cells was carried out using a NEPA21 Super Electroporator (Nepa Gene, Ichikawa, Chiba, Japan) as described previously [28] (link). For transient expression of luciferase, a mixture of 2 μg of either Nluc-reporter vector or 121-Nluc expression vector, 10 μg luc2-reference vector and 0.5 μg Tet-On 3G expression vector was transfected into the cells. One day after transfection, the medium was replaced with IPL-41 or trehalose mixture with or without doxycycline (Dox) at 1 μg/mL. The cells were collected 24 h later, and luciferase activity was measured using an ARVO luminometer (PerkinElmer, Waltham, MA) with the Nano-Glo Dual-Luciferase Reporter Assay System (Promega).
6
Quantification of Reverse Transcriptase Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with a PBS (Merck KGaA)-based lysis buffer containing 0.5% v/v Nonidet P-40, 10% w/v glycerol, 5mM DTT and Complete Mini EDTA-free Protease Inhibitor Cocktail (Merck KGaA) and sonicated for 30 s (UR-21P, TOMY SEIKO, Tokyo, Japan).
After centrifugation, aliquots of the supernatant were diluted with nuclease-free water and subjected to DNA determination using a Qubit 2.0 fluorometer and a Qubit dsDNA HS quantitation assay (Thermo Fisher Scientific). All samples were diluted to 0.436 ng/μL with lysis buffer and subjected to a reverse transcriptase assay (Thermo Fisher Scientific).
Recombinant AMV RTα (Nippon Gene, Tokyo, Japan) was used to prepare a standard curve.
The samples and Recombinant AMV RTα were diluted with lysis buffer, then mixed with a reaction mixture and incubated at 25℃. After 1 h incubation, the RT reactions were stopped by adding EDTA, and GFP fluorescence from PicoGreen was quantified using an ARVO luminometer (PerkinElmer).
After centrifugation, aliquots of the supernatant were diluted with nuclease-free water and subjected to DNA determination using a Qubit 2.0 fluorometer and a Qubit dsDNA HS quantitation assay (Thermo Fisher Scientific). All samples were diluted to 0.436 ng/μL with lysis buffer and subjected to a reverse transcriptase assay (Thermo Fisher Scientific).
Recombinant AMV RTα (Nippon Gene, Tokyo, Japan) was used to prepare a standard curve.
The samples and Recombinant AMV RTα were diluted with lysis buffer, then mixed with a reaction mixture and incubated at 25℃. After 1 h incubation, the RT reactions were stopped by adding EDTA, and GFP fluorescence from PicoGreen was quantified using an ARVO luminometer (PerkinElmer).
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!