Navigation Links
Performance of fluorescent dyes with LEADseeker Multimodality Imaging System

Key words: fluorescence • LEADseeker Cy Alexa Fluor TAMRA Brij


Flexible and scalable uHTS programs demand that organizations be able to select the most appropriate or cost-effective fluorescent reagents for the task. This application note describes experiments to assess the performance of a variety of dyes with LEADseeker™ Multimodality Imaging System. Data was collected in the system's fluorescence intensity and fluorescence polarization modes. Lowest limits of detection (LLD) were determined for the dyes using either the standard filter sets supplied with the instrument, or a collection of filter configurations defined specifically for each dye type. Results demonstrate that a variety of dyes are fully compatible with the LEADseeker Multimodality Imaging System, rendering it a versatile detection system capable of supporting a broad range of uHTS applications.


Materials
Products used

LEADseeker Multimodality Imaging System 18-1140-71

Cy™3B; Cy5 PA63101; PA15101

Alexa FluorTM 532 optical configuration 11-0013-51
(for FP modality) consists of: Fluorescence
excitation filter 500 nm, bp 40 nm Fluorescence
perpendicular dichroic Fluorescence emission
filter 568 nm, bp 50 nm

Alexa Fluor 532 Fluorescence Epi-mirror dichroic 11-0013-73

Fluorescence Epi-mirror dichroic for 11-0013-50
BODIPYTM-TMR (in FP modality)


Other materials required
Alexa Fluor 488, 532, 546, 555, and 647 (Molecular Probes)

BODIPY-TMR and BODIPY-650/665 (Molecular Probes)

Fluorescein and TAMRA™ (Molecular Probes)


Protocol
All of the fluorescent dye reagents were dissolved from powder form into DMSO at approximately 10 mg/ml. Subsequently, each dye was diluted in buffer (PBS containing 0.01% [v/v] Brij™-35) to produce solutions for determining concentration. In all cases, we determined concentration by measuring absorbance (at the absorption maxima for the dye) against a buffer-only reference and by applying the Beer Lambert equation:

i.e. c = O.D. / εl where:
c = concentration
O.D. = absorption value determined
ε = extinction coefficient of the relevant fluorescent dye

Following determination of concentration, each dye was diluted further in buffer to a top working concentration of 6.4 nM. It was then subjected to doubling dilution in buffer to produce a low concentration of 0.00625 nM. Replicates of each dilution and buffer-only reference were added (as a total volume of 50 µl per well) to a 384-well microplate and imaged (using appropriate optical components) on LEADseeker Multimodality Imaging System in either fluorescence intensity or fluorescence polarization mode for a maximum of 10 s coincidence averaging counting.


Results
Following imaging, we determined the mean value of the signal and standard deviation at each concentration for data obtained from each of the replicates in fluorescence intensity mode. LLD was then determined as the lowest dye concentration with an observed intensity value greater than that of the buffer + 3 SD of the buffer-only wells. Table 1 shows the dat a obtained for a variety of dyes using the standard optical configurations available with LEADseeker Multimodality Imaging System.

Performance was also assessed in fluorescence polarization mode. Here, too, we calculated the mean value of the signal and standard deviation at each concentration and then determined the LLD (where LLD was the lowest dye concentration with an observed polarization value consistent with that used as the reference value for that dye and with a standard deviation value of less than 10 mP for the replicates). Table 2 shows the data obtained for a variety of dyes using the standard optical configurations available on LEADseeker Multimodality Imaging System; data obtained using the dye-specific optical configurations is shown in Table 3.



Conclusion
A variety of fluorescent dyes can be used in either fluorescence intensity or fluorescence polarization mode with LEADseeker Multimodality Imaging System.

The ability to choose from standard filter sets or from dyespecific optical configurations enhances the flexibility of LEADseeker Multimodality Imaging System.



back to top
'"/>

Source:


Page: All 1 2 3

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:
*Name:
*Comment:
*Email:


(Date:5/25/2016)... ... May 25, 2016 , ... Thailand’s Board of Investment’s ... in San Francisco. Located at booth number 7301, representatives from the Thai Government, ... discuss the Thai biotechnology and life sciences sector. , Deputy Secretary General ...
(Date:5/25/2016)... ... ... Scientists at the University of Athens say they have evidence that the variety ... that could lead to one good one. Surviving Mesothelioma has just posted an article ... team evaluated 98 mesothelioma patients who got a second kind of drug ...
(Date:5/24/2016)... , ... May 24, 2016 , ... ... diabetes, and traumatic injuries, will be accelerated by research at Worcester Polytechnic Institute ... engines of wound healing and tissue regeneration. , The novel method, developed by ...
(Date:5/23/2016)... ... ... The need for blood donations in South Texas and across the nation is growing. , ... Center, blood donations are on the decline. In fact, donations across the country are at ... South Texas in the last four years alone. , There is no substitute for blood. ...
Breaking Biology Technology:
(Date:3/9/2016)... , March 9, 2016 This BCC Research ... states of the RNA Sequencing (RNA Seq) market for ... as instruments, tools and reagents, data analysis, and services. ... segments of the RNA-Sequencing market such as RNA-Sequencing tools ... the main factors affecting each segment and forecast their ...
(Date:3/9/2016)... GARDENS, Fla. , March 9, 2016 /PRNewswire/ ... management authentication and enrollment solutions, today announced the ... DigitalPersona ® Altus multi-factor authentication platform. ... and InfoSec managers to step-up security where it,s ... Washington, DC . ...
(Date:3/3/2016)...  FlexTech, a SEMI Strategic Association Partner, awarded five ... Development, Leadership in Education, and, in a category new ... year of the FLEXI Awards and the winners ... past years . Judging was done on a ... criteria, by a panel of non-affiliated, independent, industry experts. ...
Breaking Biology News(10 mins):