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View the relevant questions below:

Luciferase Reporter Assays

The problems with white plates are related to cross-talk blocking, surface reflectivity and tendency to phosphorescence. We do not recommend using white plates because they give much higher background even if adapted to darkness for 10 minutes. The white plastic does not block the light completely, and some portion of the signal will go through the plastic and will be seen when you measure adjacent wells. The typical situation is if you change from black to white plates, where the background is about doubled and the signal is increased about 10 times. Black plates are recommend for best signal to noise ratio even though the RLU values will be lower. 

For Fetal Bovine Serum (FBS), there is no difference between 10% and 5% FBS. It is worthwhile to note that the type of serum does affect the luciferase assay. We tested the effect of 6 different types of sera (FBS, heat-inactivated FBS, dialyzed FBS, charcoal-treated FBS, donor adult bovine serum, and donor equine serum) on secreted luciferases, e.g. Gaussia, Gaussia-Dura, and Cypridina Luciferases. No noticeable difference was observed among different types of sera except for donor adult bovine serum. Unknown factors in donor adult bovine serum caused up to 35% inhibition in secreted luciferase activity. Therefore, we don’t recommend using donor adult bovine serum for Gaussia, Gaussia-Dura, and Cypridina Luciferases.

All Luciferase Glow Assay Working Solutions should be stored protected from light.

  • Renilla Luciferase Glow Assay Working Solution must be stored at room temperature (20-25°C) before use and is stable for up to 8 hours at room temperature.
  • Gaussia Luciferase Glow Assay Working Solution must be stored at room temperature (20-25°C) before use and is stable for up to 4 hours at room temperature. 
  • Cypridina Luciferase Glow Assay Working Solution must be stored at room temperature (20-25°C) before use and is stable for up to 2 hours at room temperature.
  • Firefly Luciferase Glow Assay Working Solution must be stored at room temperature (20-25°C) before use and is stable for 4 hours at room temperature. For long term use, store at -20 degrees C for up to 2 months.

Here are possible causes and solutions:

Causes

Solution

 

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°C; maintain 100X Vargulin at -80°C 

Prepare new Coelenterazine Working Solution if used longer than 8 hours; prepare new Vargulin Working Solution if used longer than 2 hours

Here are possible causes and solutions:

Causes

Solution
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 

Here are possible causes and solutions:

Causes

Solution
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 

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.

Here are possible causes and solutions:

Causes

Solution
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

Here are possible causes and solutions:

Causes

Solution
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°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 

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 
    Note: A low sample volume can increase assay variability. Dilute the sample and use the recommended volume of 10-20 μL per assay.

This could be due to contamination of the control sample. Make sure to use new sample and change pipette tips after each well.

Here are possible causes and solutions:

Causes

Solution
Low transfection efficiency 

Optimize transfection conditions using a visual transfection control (e.g., a plasmid over-expressing a fluorescent protein) 

Verify plasmid DNA quality using restriction digestion and agarose gel electrophoresis; use only transfection grade DNA 

Note: Most high-quality plasmid DNA should be supercoiled

Use actively dividing, low-passage cells 

Use a different cell type 

No promoter induction

Incubate cells using promoter-specific inducing conditions

Incubate cells for a longer time after treatment

Change growth conditions to improve expression 

Use a different promoter 

Substrate auto-oxidized 

Protect substrate from light and air

Maintain 100X Coelenterazine at -80°C, 100X Vargulin at -20°C, and 100X D-Luciferin at -20°C,

Here is a possible cause and its solutions:

Cause

Solutions
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 

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: 
    Note: A low sample volume can increase assay variability. Dilute the sample and use the recommended volume of 10-20 μL per assay.

Here are possible causes and solutions:

Causes

Solution

Non-specific oxidation of 

substrate

Use new sample

Avoid repeated freezing and thawing of the sample

Control sample is contaminated

Change pipette tips after each well

Reduce shaker speed during the cell lysis step to avoid contaminating the wells 

Here are possible causes and solutions:

Causes

Solution
Low transfection efficiency 

Optimize transfection conditions using a visual transfection control (e.g., a plasmid over-expressing a fluorescent protein) 

Verify plasmid DNA quality using restriction digestion and agarose gel electrophoresis; use only transfection grade DNA 

Note: Most high-quality plasmid DNA should be supercoiled

Use actively dividing, low passage cells 

Use a different cell type 

No promoter induction

Incubate cells under promoter-specific inducing conditions 

Incubate the cells for a longer time after treatment

Change growth conditions to improve expression 

Use a different promoter or cell type

TurboLuc™ One-Step Substrate auto-oxidized 

Protect substrate from light and air and store at -80°C 

Prepare Working Solution immediately before use and protect from light

Wrong substrate used

Use only substrate supplied with the kit. Coelenterazine from related Luciferase kits (e.g., Renilla Luciferase) will provide suboptimal performance 

Low luciferase expression

Use a different promoter or growth conditions to improve expression 

Adjust instrument parameters to capture more signal (instrument-dependent) 

Plate larger number of cells 

Here are possible causes and solutions:

Causes

Solution
High luciferase expression 

Reduce incubation time before collecting samples 

Adjust instrument parameters to capture less signal (instrument-dependent) 

Decrease the amount of plasmid transfected into cells or decrease cell number 

Control sample contaminated   Change pipette tips after each well 

beta-Galactosidase Assay Kits

Using the Assay Stop Solution is optional, however the absorbance needs to be read immediately on the plate reader after the 30 minute incubation when the Stop Solution is not added to the assay, due to a shift in absorbance over time.