Navigation Links
Use of the HitHunter EFC Chemiluminescent Detection Kits with LEADseeker Multimodality Imaging System

Key words: EFC chemiluminescence • HitHunter LEADseeker


Competitive pressure is forcing organizations to continually increase screening throughput. Yet accuracy and sensitivity remain vital concerns, particularly for common screens such as luminescence assays. LEADseeker™ Multimodality Imaging System was developed to meet these demands by combining excellent detection with the ability to image whole plates in less than one minute.

This application note describes the use of the HitHunter™ range of EFC chemiluminescent assays with the LEADseeker Multimodality Imaging System. The Caspase 3 and p38 MAPK binding assay kits were used as model systems. The data demonstrate that the HitHunter range of assay kits, which are based on enzyme fragment complementation technology, perform well in conjunction with the chemiluminescent readout of LEADseeker Multimodality Imaging System equipped with the supplied luminescence filter. Used together, the components support organizations interested in adding high and ultrahigh throughput chemiluminescent assays to their screening programs.

Materials
Products used

LEADseeker Multimodality Imaging System 18-1140-71

HitHunter Caspase 3 EFC Chemiluminescent 90-0028-02
Detection Kit

HitHunter p38 MAPK Binding Assay EFC 90-0028-31(L)
Chemiluminescent Detection Kit


Other materials required
Luminescence 660 nm cut-off filter

384-well plates (non-treated, non-sterile, polystyrene, white)

DEVD-CHO inhibitor (BIOMOL)

SB203580 and SB239063 inhibitors (Calbiochem)


Method
1. Caspase 3 method
(Note: The assay was set up in triplicate and all reagents werehawed on ice. The assay was carried out at room temperature.)

1.1 Prepare all reagents as recommended by the kit manufacturer.

1.2 Prepare Caspase 3 enzyme standard curve working dilutions in assay buffer.

1.3 Prepare DEVD-CHO inhibitor curve working dilutions in assay buffer.

1.4 Create "No Protease" control wells by adding 15 µl assay buffer to appropriate wells.

1.5 Add 5 µl assay buffer to all enzyme standard curve wells.

1.6 Create Caspase 3 standard curve by adding 10 µl from the working dilutions (see 1.2 above) to appropriate wells (final concentration range = 0.4–100 pg/well)

1.7 Add 10 µl Caspase 3 enzyme from the appropriate working dilution (see 1.2 above) to all inhibitor curve wells (final concentration = 100 pg/well).

1.8 Create DEVD-CHO inhibitor curve by adding 5 µl from the working dilution (see 1.3 above) to appropriate wells (final concentration range = 0.1–100 000 nM).

1.9 Add 5 µl ED Reagent to all wells and incubate the assay 3 h at room temperature.

1.10 Add 15 µl EA Reagent and 15 µl CL Substrate to all wells and incubate the assay 1 h at room temperature.

1.11 Image the plates for 30 s on Leadseeker Multimodality Imaging System. (Note: Due to the excellent sensitivity of LEADseeker Multimodality Imaging System, a reduced imaging time of 30 s is recommended to achieve the same results as described in the pack leaflet.)

2. p38 MAPK method
(Note: The assay was set up in triplicate and all reagents were thawed on ice. The assay was carried out at room temperature.)

2.1 Prepare all reagents as recommended by the kit manufacturer.

2.2 Create "No inhibitor" control wells by adding 20 µ l assay buffer to appropriate wells.

2.3 Create inhibitor curve wells by adding 20 µl SB 203580 or SB239063 inhibitor diluted in assay buffer to appropriate wells (final concentration range = 0.045–2650 nM)

2.4 Add 10 µl p38/EA Reagent mix to all the wells and incubate the assay 30 min at room temperature.

2.5 Add 10 µl ED Reagent to all wells and incubate the assay 30 min at room temperature.

2.6 Add 10 µl CL Substrate to all wells and incubate the assay 1 h at room temperature.

2.7 Image the plate on LEADseeker Multimodality Imaging System for 30 s. (Note: Due to the excellent sensitivity of LEADseeker Multimodality Imaging System, a reduced imaging time of 30 s is recommended to achieve the same results as described in the pack leaflet.)


Results
Caspase 3
The standard curves generated in two separate assays (Fig 1) were entirely consistent and comparable with the pack leaflet data. Both assay curves also gave excellent r2 values (0.99 and 0.96 for assay 1 and 2 respectively).

The IC50 results generated (Fig 2) were also consistent with the pack leaflet data. The value of 1.8 nM obtained was within 95% confidence intervals of the pack leaflet value (1.54 nM).

Z` data was generated by using the top standard (10 ng per well) as the positive control and the no-protease control as the negative control. Z` results are shown in Figure 3. The calculated Z` value of 0.67 confirms that the Caspase 3 assay is a suitable format for use with the LEADseeker Multimodality Imaging System.

p38 MAPK binding assay
The IC50 results generated for this assay (Fig 4) were consistent and comparable with the pack leaflet data. The values of 32 nM and 37 nM for the inhibitors SB203580 and SB239063, respectiv ely, were within 95% confidence intervals of the pack leaflet values (29 nM and 33 nM, respectively).


Conclusion
HitHunter Caspase 3 and p38 chemiluminescent assays are fully compatible with LEADseeker Multimodality Imaging System.

Enzyme titration, inhibition assay, and Z` analyses all demonstrate that these assays perform to pack leaflet specifications on LEADseeker Multimodality Imaging System.

LEADseeker Multimodality Imaging System and HitHunter assay kits can be effectively used in combination by organizations seeking to run high and ultra-high throughput luminescence assays.




back to top
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Performance of the AMARA HitHunter Chemiluminescence Kinase Assay with the LEADseeker Multimodality Imaging System
2. Imaging Chemiluminescent Western Blots
3. Quantitative Measurement of Cell Proliferation Using the BrdU ELISA: A Comparison Between Colorimetric and Chemiluminescent Detection
4. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
5. A New PCR-based Mycoplasma Detection Method
6. HSVision Molecular Beacon Detection Module Rapidly Detects Herpes Simplex Virus DNA
7. Detection and Identification of Phosphorylation Sites in Proteins Using LC/MS/MS with Neutral Fragment Loss Mapping
8. Detection of mRNAs on Cryosections of the Cardiovascular System Using DIG-Labeled RNA Probes
9. Gene Expression Arrays: Highly Sensitive Detection of Expression Patterns with Improved Tools for Target Amplification
10. The DIG System Nonradioactive and Highly Sensitive Detection of Nucleic Acids
11. Quantification of Nucleosomes in Serum by the Cell Death Detection ELISAplus
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... , June 27, 2016  Alex,s Lemonade Stand ... that that it will open a state-of-the-art bioinformatics lab, ... cancer research. This announcement comes as Liz Scott ... the National Cancer Moonshot Summit in Washington, ... Biden as a participant and advocate of pediatric cancer ...
(Date:6/27/2016)...  Global demand for enzymes is forecast to ... $7.2 billion.  This market includes enzymes used in ... production, animal feed, and other markets) and specialty ... and beverages will remain the largest market for ... products containing enzymes in developing regions.  These and ...
(Date:6/27/2016)... /PRNewswire/ - BIOREM Inc. (TSX-V: BRM) ("Biorem" or "the Company") ... shareholders, Clean Technology Fund I, LP and Clean Technology ... based venture capital funds which together hold approximately 59% ... diluted, as converted basis), that they have entered into ... holdings in Biorem to TUS Holdings Co. Ltd. ("TUS") ...
(Date:6/27/2016)... , June 27, 2016  Sequenom, Inc. (NASDAQ: ... to enabling healthier lives through the development of innovative ... of the United States denied ... that the claims of Sequenom,s U.S. Patent No. 6,258,540 ... eligibility criteria established by the Supreme Court,s Mayo Collaborative ...
Breaking Biology Technology:
(Date:6/27/2016)... DUBLIN , June 27, 2016 Research ... in North America 2016-2020" report to their offering. ... North America to grow at a CAGR ... has been prepared based on an in-depth market analysis with inputs ... growth prospects over the coming years. The report also includes a ...
(Date:6/22/2016)... 22, 2016  The American College of Medical Genetics and ... Magazine as one of the fastest-growing trade shows during ... the Bellagio in Las Vegas . ... of growth in each of the following categories: net square ... number of attendees. The 2015 ACMG Annual Meeting was ranked ...
(Date:6/22/2016)... , June 22, 2016   Acuant ... and verification solutions, has partnered with RightCrowd ... solutions for Visitor Management, Self-Service Kiosks and ... products that add functional enhancements to existing ... corporations and venues with an automated ID ...
Breaking Biology News(10 mins):