What could be causing no signal or low signal with the Firefly Luciferase Glow/Luciferase Flash assay? Do you have any suggestions to get around this problem?
Here are possible causes and solutions:
- Low transfection efficiency: Optimize transfection conditions using a visual transfection control (e.g., a plasmid over-expressing a fluorescent protein); Verify plasmid DNA quality - use only transfection grade DNA; Use actively dividing, low passage cells; Use a different cell type.
- No or low promoter activity: Use conditions known for promoter activation; Incubate cells for a longer time; Change growth conditions to improve expression; Use a different promoter.
- D-luciferin auto-oxidized: Protect substrate from light and maintain 100X D-luciferin at -20 degrees C; Prepare new Working Solution if used longer than 4 hours.
- Low luciferase expression: Use Pierce Firefly Signal Enhancer (100X) (Cat. No. 16180); Lyse cells in a smaller volume of 1X Cell Lysis Buffer; Use a different promoter or growth conditions to improve expression; Increase the integration time on the instrument; Scale-up the volume of sample and reagent per well.
- Degraded luciferase protein: Store cell lysates on ice and perform assays immediately following cell lysis.
I got a high background signal with a Gaussia/Cypridina/Renilla Luciferase Glow/Luciferase Flash assay. What went wrong?
Here are possible causes and solutions:
- Non-specific oxidation of substrate: Use less serum in the cell culture media; Note: Albumin can increase the auto-oxidation of Vargulin; Avoid repeated freezing and thawing of the sample.
- Control sample is contaminated: Use new sample; Change pipette tips after each well.
I am getting a high saturating signal with a Gaussia/Cypridina/Renilla Luciferase Glow/Luciferase Flash assay. What could have happened?
This could be due to high luciferase expression. Here are some suggestions:
Reduce incubation time before collecting samples.
Decrease the integration time on the instrument.
Dilute the sample: for secreted Gaussia/Cypridina Luciferase, dilute the sample using media from the cell culture; for cell lysate, dilute the sample using lysis buffer
Note: A low sample volume can increase assay variability. Dilute the sample and use the recommended volume of 10-20 µL per assay.