UA-Glo® cAMP Reagent
UA-Glo® cAMP Reagent
Product Details
Product Details
Product Specification
| Stability & Storage | Dry ice transportation. Store protected from light at -80°C. Upon initial use, prepare as UA-Glo® cAMP reagent stock solution, then aliquot as needed and store protected from light at -80°C, avoiding repeated freeze-thaw cycles. |
Background
The UA-Glo® cAMP reagent is suitable for measuring changes in cAMP concentration in live cells using the Promega GloSensor™ cAMP assay system, with detection performance comparable to that of the Promega GloSensor™ cAMP Reagent (Cat E1290, E1291).
Components
The components and specifications of UA-Glo® cAMP reagent are as follows. The detectable number of wells in 96-/384-well plates is calculated based on the standard experimental conditions where 100/20 μL of balanced medium is added per well in 96-/384-well plates, respectively.
cAMP Reagent |
HEPES Buffer (10mM, pH7.5) |
Detectable 96-Well Plate Wells |
Detectable 384-Well Plate Wells |
25 mg |
1 mL |
2,000 |
|
250 mg |
10 mL |
20,000 |
Protocol
According to the Promega GloSensor™ cAMP assay system manual (TM076), design and perform relevant experiments. Below are the experimental steps for cAMP detection in adherent cells using UA-Glo® cAMP reagent.
1)Prepare UA-Glo® cAMP reagent stock solution: Add 25mg UA-Glo® cAMP reagent (L7101) to 817μL of 10mM HEPES buffer (pH7.5), or add 250mg UA-Glo® cAMP reagent (L7102) to 8.17mL HEPES buffer (pH7.5). Mix thoroughly. Aliquot as needed and store protected from light at -80℃, avoiding repeated freeze-thaw cycles. This cAMP reagent stock solution can be diluted 50x as described in step 3 to prepare equilibration medium, or the concentration of cAMP reagent in the equilibration medium can be optimized for maximum signal-to-background difference.
2)Prepare equilibration medium aseptically as needed:
88% CO2-independent medium (e.g., Invitrogen Cat.#18045 )
10% fetal bovine serum
2% UA-Glo® cAMP reagent stock solution
3)Remove the 96-well experimental cell plate (e.g., cells expressing Promega GloSensor™ or transiently transfected cells), carefully aspirate the medium from the wells, and gently add 100μL of the equilibration medium prepared in step 2. Incubate at room temperature (22-25℃) for 2hr. The incubation time and temperature can be optimized based on when background signals stabilize. Ensure consistent temperature across the plate during detection.
4)Add test compounds and appropriate controls according to the experimental design. For example, use 10μM forskolin as a positive control and the solvent vehicle as a negative control.
5)Measure luminescence signals using a luminometer or multifunctional plate reader 15-30min after compound addition. Refer to section 3.A of the Promega GloSensor™ cAMP assay system manual (TM076) for specific timing or optimize based on the experiment.
Guidelines
