Screen Quest™ No Wash Potassium Channel Assay Kit
Price | |
Catalog Number | |
Unit Size | |
Quantity |
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Correction Factor (260 nm) | 1.076 |
Correction Factor (280 nm) | 0.769 |
Extinction coefficient (cm -1 M -1) | 23430 |
Excitation (nm) | 495 |
Emission (nm) | 516 |
Quantum yield | 0.161 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | SDSProtocol |
Correction Factor (260 nm) 1.076 | Correction Factor (280 nm) 0.769 | Extinction coefficient (cm -1 M -1) 23430 | Excitation (nm) 495 | Emission (nm) 516 | Quantum yield 0.161 |
Platform
Fluorescence microplate reader
Excitation | 490 nm |
Emission | 525 nm |
Cutoff | 515 nm |
Recommended plate | Black wall/clear bottom |
Instrument specification(s) | Bottom read mode/Programmable liquid handling |
Other instruments
ArrayScan, FDSS, FLIPR, FlexStation, IN Cell Analyzer, NOVOStar, ViewLuxComponents
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells in growth medium
- Add Tl-520 AM dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
- Incubate at 37°C for 1 hour
- Add agonist/antagonist and incubate at 37 °C for 30 minutes
- Add Tl2SO4/K2SO4 Stimulus solution (50 µL/well for 96-well plate or 12.5 µL/well for 384-well plate)
- Monitor fluorescence intensity at Ex/Em = 490/525 nm
Important notes
Do not add additional probenecid. Thaw all the kit components at room temperature before use.
PREPARATION OF STOCK SOLUTION
1. TI-520 AM stock solution:
Add 20 µL (Cat. # 36550) or 200 µL of (Cat. # 36551 and # 36552) of DMSO into the vial of TI-520 AM (Component A), and mix them well. Note: The unused TI-520 AM stock solution can be aliquoted and stored at < -20 oC. Protect from light and avoid repeated freeze-thaw cycles.
2. Assay Buffer (1X):
Add 1 mL of 10X Pluronic® F127 Plus (Component B) into 9 mL of HHBS buffer (Component C) and mix well. Note: 10 mL of 1X assay buffer is enough for one plate. Aliquot and store un-used 10X Pluronic® F127 Plus (Component B) at < -20 oC. Protect from light and avoid repeated freeze-thaw cycles.
3. Chloride Free Buffer (1X):
Add 2 mL of Chloride Free Buffer (5X) (Component D) in 8 mL of ddH2O, and mix them well.
4. Tl2SO4 solution (Not provided, Sigma Cat# 208191):
Dissolve Tl2SO4 in ultrapure H2O to final concentration of 80 mM. Note: Tl2SO4 is toxic. Take necessary precautions to prevent inhalation and skin contact.
PREPARATION OF WORKING SOLUTION
1. TI-520 AM dye-loading solution:
Add 20 µL of TI-520 AM stock solution (500X) into 10 mL of 1X Assay Buffer and mix well.
2. Stimulus Solution(5X):
Dilute ligands (for non-voltage gated potassium channels) or K2SO4 (for voltage gated potassium channels) with Tl2SO4 in 1X Chloride-free buffer.
Table 1. Reference (for voltage gated hERG channel in HEK293-KCNH2 cells), concentration of Tl2SO4 and K2SO4 (for voltage gated potassium channels) or ligands (for non-voltage gated potassium channels) used for the assay should be optimized for each target channel and cell type.
Components |
Volume |
5X Conc |
Final Conc |
Chloride-free Buffer(5X) |
2 mL |
|
|
K2SO4 (250 mM) |
1 mL |
25 mM |
5 mM |
TI2SO4 (80mM) |
0.5 mL |
4 mM |
0.8 mM |
ddH2O |
5.5 mL |
|
|
Total: |
10 mL |
|
|
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of TI-520 AM dye-loading solution into the cell plate.
- Incubate the dye-loading plate in a cell incubator for 1 hour, and then incubate the plate at room temperature for another 15 to 30 minutes. Note: If the assay requires 37°C, perform the experiment immediately without further room temperature incubation.
- Prepare the K+ channel antagonists or agonists with HHBS.
- Incubate the plate with antagonists (for inhibitory study) in the cell incubator for 30 minutes.
- Add 50 µL/well (for a 96-well plate) or 12.5 µL/well (for a 384-well plate) of 5X stimulus buffer with FLIPR, FDSS or Flexstation. Run the experiment with a filter set of Ex/Em = 490/525 nm. Read the plate every 1–2 seconds for 3 minutes.
Spectrum
Spectral properties
Correction Factor (260 nm) | 1.076 |
Correction Factor (280 nm) | 0.769 |
Extinction coefficient (cm -1 M -1) | 23430 |
Excitation (nm) | 495 |
Emission (nm) | 516 |
Quantum yield | 0.161 |
Images
Application notes
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