Navigation Links
Performance of the AMARA HitHunter Chemiluminescence Kinase Assay with the LEADseeker Multimodality Imaging System

Key words: AMARA • kinase assay • LEADseeker • chemiluminescence


It is predicted that at least 500 kinase and phosphatase enzymes exist, their activities having wide-ranging regulatory responses. Many kinases are part of a signaling cascade in disease pathways such as inflammation and cancer, so identifying modulators of kinase and phosphatase activity may provide new therapeutic agents.

This application note provides recommendations for the successful imaging of the AMARA HitHunter™ Chemiluminescence Kinase Assay on the LEADseeker™ Multimodality Imaging System and demonstrates the assay’s ability to measure the functional activity of serine/threonine kinase AMP in the phosphorylation of the AMARA substrate (AMARAASAAALARRR).


Materials
Reagents and instrumentation

AMARA HitHunter Chemiluminescence 90-0060-02
Kinase Assay Kit*, 800 (384 wells)

LEADseeker Multimodality Imaging System 18-1140-71

[14C] Uniform Reference Plate CFQ11387-4UCI


Additional materials required
AMP-activated protein kinase (Upstate #14-305)

ATP (Sigma A-7699)

Adenosine monophosphate (AMP) (Sigma A-2002)

384-well white flat bottom polystyrene (Corning 3705)
not treated microplates

Graphpad Prism software v.4 (GraphPad Software)


Method
LEADseeker setup
A flat field correction was established using a [14C] Uniform Reference Plate. No background correction was required.

Plates were imaged on the LEADseeker Multimodality Imaging System for 60 s with coincident averaging and 3 × 3 binning.

Reagent preparation
1. The kinase enzyme was diluted using the antibody reagent supplied in the kit to give a range of working stocks yielding a final concentration range in the assay from 0.05 to 100 units/well.

2. The AMARA substrate and ATP were diluted with the assay buffer supplied in the kit to yield working stocks of 20-µM AMARA substrate, 40-µM ATP, and 400-µM AMP.

3. The chemiluminescent substrate (CLS) was prepared by mixing 1 part Galacton-Star™, 5 parts Emerald-II™, and 19 parts CLS diluent.

4. The enzyme donor (ED)/CLS mix was prepared by carefully mixing 1 part ED reagent with 1 part CLS and was used within 6 h.

5. The enzyme acceptor (EA) reagent was used as supplied.

Assay procedure

1. To the kinase assay wells, add 10 µl of kinase reaction buffer to each well. To the standard curve wells, add 10 µl of AMARA standard and 10 µl of AMARA antibody reagent.

2. Incubate plate in darkness for 60 min at room temperature (20–25 ºC).

3. Add 20 µl of ED/CLS to each well.

4. Incubate plate in darkness for 60 min (± 15 min) at room temperature.

5. Add 10 µl of EA reagent to each well.

6. Incubate plate in darkness at room temperature for at least 60 min or overnight.

7. Seal plate and image on the LEADseeker Multimodality Imaging System for between 10 and 60 s.

Preparation of standard curve
The standard curve was established by preparing twelve 3-fold serial dilutions (using assay buffer) of the phosphorylated AMARA standard supplied in the kit. The final concentration range of the standard curve was 5.6 × 10-11 to 1.0 × 10-5 M in a final volume of 20 µl/well. Each standard concentration was assayed in triplicate.

AMP kinase titration procedure
AMP kinase was diluted in antibody reagent to yield a final concentration range of 0.05 to 100 mU/well. Each test point was assayed in triplicate. The AMARA substrate was used at a final concentration of 10 µM; ATP was used at a final concentration of 20 µM and AMP at 200 µM. The reaction was incubated for 60 min at room temperature. Following incubation with the detection reagents, the reaction was imaged on the LEADseeker Multimodality Imaging system for 60 s.


Conclusion
The AMARA HitHunter Chemiluminescence Kinase Assay has been successfully used on the LEADseeker Multimodality Imaging System and is suitable for measuring the phosphorylation of AMARA substrate by serine/threonine kinase AMP.


* The assay buffer may require supplementation with cofactors, which will be enzyme specific. This application note describes how the AMARA HitHunter™ Chemiluminescence Kinase Assay performs with serine/threonine kinase AMP; no additional cofactors were added to the assay buffer for this enzyme; however, there is a 3-fold increase in activation of the enzyme in the presence of adenosine 5’-monophosphate (AMP).

† Other pack sizes available, please inquire for details.



back to top
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Novel PCR Enhancing Factor Improves Performance of Pfu DNA Polymerase
2. Comparing Fidelity and Performance of Proofreading PCR Enzymes
3. PCR Performance Comparisons Between pfuturbo and Taq DNA Polymerases
4. pfuturbo DNA Polymerase: A High-Performance, High-Fidelity Enzyme Ideal for PCR Cloning
5. Performance Comparisons of Commercial RT-PCR Systems
6. Restriction Endonucleases Undergo Rigorous QC Testing to Ensure Optimal Performance
7. Unique Enhanced DNA Polymerase Delivers High Fidelity and Great PCR Performance
8. Versatile Transfection Reagent Offers Low Toxicity and Consistent Performance
9. Challenge the Performance of Your Hot-Start PCRs with FastStart Taq DNA Polymerase and the Novel FastStart High Fidelity PCR System
10. Highly Sensitive microRNA Array Performance
11. High Throughput RNA Isolation for High Performance Microarray Analysis
Post Your Comments:
(Date:8/20/2014)... High performance computing (HPC) will ... complete climate and Earth system model to address ... , Eight national laboratories, including Lawrence Livermore ... for Atmospheric Research, four academic institutions and one ... national laboratories include Argonne, Brookhaven, Lawrence Berkeley, Los ...
(Date:8/20/2014)... , Aug. 20, 2014  PAREXEL International Corporation ... will be presenting at the Baird Healthcare Conference in ... and Chief Financial Officer, will be making a presentation ... EDT on Wednesday, Sept. 3, 2014. A ... the "Investors" section of PAREXEL,s website at www.PAREXEL.com ...
(Date:8/20/2014)... Toronto, Canada (PRWEB) August 20, 2014 ... wisdom that E&L studies should be performed late in ... system is known. But current regulatory trends suggest that, ... are on the rise. In recent years, multiple drug ... E&L packages or address specific questions during phase I/II. ...
(Date:8/20/2014)... your ear could improve the health of your heart, ... of Leeds used a standard TENS machine like those ... to the tragus, the small raised flap at the ... ear canal. , The stimulation changed the influence of ... nervous signals that can drive failing hearts too hard. ...
Breaking Biology Technology:New Project Is the ACME of Addressing Climate Change 2New Project Is the ACME of Addressing Climate Change 3PAREXEL International To Present At Baird Healthcare Conference 2Are Extractable and Leachables going Phase-Appropriate? New Webinar Hosted by Xtalks 2'Tickling' your ear could be good for your heart 2
... Abstract , ... become a standard for many biological assays such as high ... high volume procedures has been limited by the amount of ... microplate assays can be efficiently counted in microplates with the ...
... The DELFIA time-resolved fluorescence assay for inhibition of poly(ADP-ribose) ... the inhibition of PARP-activated incorporation of biotinylated nicotinamide adenine ... The method is a convenient way to screen test ... and secondary screening. , ...
... applications, in optoelectronics, ... and sources of laser light. For example, alloyed CdSeTe QDs are shown, ... a nonlinear change in their photoluminescence spectra, ... Spex ...
Cached Biology Technology:Counting Non-Aqueous Samples in the TopCount Microplate Scintillation and Luminescence Counter 2Counting Non-Aqueous Samples in the TopCount Microplate Scintillation and Luminescence Counter 3Counting Non-Aqueous Samples in the TopCount Microplate Scintillation and Luminescence Counter 4Counting Non-Aqueous Samples in the TopCount Microplate Scintillation and Luminescence Counter 5A DELFIA Assay for Inhibition of PARP 2A DELFIA Assay for Inhibition of PARP 3Photoluminescence Spectroscopy of Quantum Dots, monitored by the Spex FluoroMax 2Photoluminescence Spectroscopy of Quantum Dots, monitored by the Spex FluoroMax 3Photoluminescence Spectroscopy of Quantum Dots, monitored by the Spex FluoroMax 4
(Date:8/20/2014)... , Aug. 20, 2014 /CNW/ -  Issue ... and can quickly become life threatening for some children.    ... as 1.2 million Canadians have food allergies and this number ... cent of children are estimated to have food allergies.  ... mild skin irritations and hives to breathing difficulties and loss ...
(Date:8/20/2014)... Scientists from the University of Tbingen, Arizona State University, ... and Public Health Institute (Swiss TPH) isolated Mycobacterium ... at least 1000 years old. The pathogen is a ... only occasionally causes disease in humans today. These researchers ... the Peruvian coast. "The link to sea lions was ...
(Date:8/20/2014)... visible in in this image of the Para and ... were most likely intentionally set in order to deforest ... or stand of trees where the land is thereafter ... conversion of forestland to farms, ranches, or urban use. ... of the Amazon Rainforest in the middle of the ...
Breaking Biology News(10 mins):Information Update - Allergens and food safety at school - What's in your child's lunch? 2Information Update - Allergens and food safety at school - What's in your child's lunch? 3
... contribute to the greenhouse gas emissions that are causing climate change. ... How we travel, what we eat, what we consume and what ... our carbon footprint . How, though, do we measure ... to find out on April 26, when Louis S. Santiago ...
... children in the United States are obese, and in inner-city ... diets and physical inactivity are the main culprits, there is ... A study by Columbia University,s Mailman School of Public ... to higher concentrations of chemicals called polycyclic aromatic hydrocarbons, or ...
... of the most common types of human leukemia may ... to new research led by physician-scientists at the University ... Cancer Center. Published this week in the journal ... in the FLT3 gene as targets for acute myeloid ...
Cached Biology News:Measuring our carbon footprint 2Prenatal exposure to air pollution linked to childhood obesity 2Prenatal exposure to air pollution linked to childhood obesity 3An Achilles heel of AML 2An Achilles heel of AML 3
... routine agarose gel electrophoresis into an automated, ... This bufferless, pre-cast system is ideal for ... digests, and more. Fully automated, robot-compatible, and ... 96 system makes your high-throughput screening assignments ...
SpectroQuest single beam 4nm UV/Vis spectrophotometer; stand-alone unit; large angled LCD screen; large sample compartment...
... PCR Cloning Kit, 40 reactions. Optimized ... of all PCR products with blunt ... approaches by offering consistently better signal-to-noise ... more positive clones.Amplicons generated by proof-reading ...
... Clone/PAD: ZMD.368. Immunogen: Synthetic ... of the human WNT2B/WNT13 protein. ... human mouse and rat WNT2B/WNT13 proteins. ... isoforms. Reactivity: Mouse Human Rat ...
Biology Products: