Pierce™ Renilla Luciferase Flash Assay Kit

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

This could be due to high luciferase expression. Here are some suggestions:

Here are possible causes and solutions:

- Non-optimized lysis buffer used: Assay luciferase activity in the media to confirm good expression of luciferase; Use only the provided lysis buffer.
- Low luciferase expression: Lyse cells in 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 volume of sample and reagent per well.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Here are possible causes and solutions:

- Insufficient luciferase accumulation in media: Incubate cells for a longer time.
- Low luciferase expression: Use less media per well during the experiment;.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.
- Treatment interfered with cellular secretory pathway: Transfect cells with a plasmid for constitutive expression of Luciferase (i.e., with pTK or pCMV promoter); Determine if luciferase actively expresses in media without treatment. Add treatment; Determine if there is a corresponding drop in luciferase activity from the constitutively expressed plasmid.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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.
- Substrate auto-oxidized: Protect substrate from light and air; Maintain 100X Coelenterazine at -80 degrees C; maintain 100X Vargulin at -80 degrees C; Prepare new Coelenterazine Working Solution if used longer than 8 hours; Prepare new Vargulin Working Solution if used longer than 2 hours

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.