Gaussia Luciferase Cellular Assay

Transcription

1 Product Guide Gaussia Luciferase Cellular Assay Gaussia Luciferase Assay Kits Cat # ,000 assays Cat # ,000 assays

2 Table of Contents: Contents of GLum.1, Gaussia Luciferase Assay Kit:... 2 Storage of Assay Kit Reagents:... 2 Overview:... 3 GLum.1 Advantages... 3 GLum.1 Gaussia Luciferase Assay Kit Protocols:... 4 I. Secreted Gaussia Luciferase (sgl.1):... 4 II. Destabilized, Membrane-Anchored, Gaussia Luciferase (mgl.1):... 5 Application Notes:...7 References:... 8 Contents of GLum.1, Gaussia Luciferase Assay Kit: # A # A GLum.1, Substrate ul.. 5 ml GLum.1, Assay Buffer (sterile).. 2 x 50 ml.. 5 x 200 ml Storage of Assay Kit Reagents: Storage Shelf Life GLum.1, Substrate:.-20 o C or -80 o C (dark)..6 months GLum.1, Assay Buffer (sterile).4 o C (keep sterile)...6 months 2

3 Overview: GLum.1, Gaussia Luciferase Assay Kit from Xactagen is designed to provide fast and sensitive measurement of luciferase from Gaussia princeps (a marine copepod). 1,2,3 Gaussia luciferase catalizes oxidation of coelenterazine in a ATP independent reaction that emits light and CO 2 (Figure 1). Humanized enzyme has been shown to have up to 1,000 fold more activity than firefly or Renilla luciferase and is used both in vitro and in vivo when combined with whole-animal imaging). Two forms of Gaussia luciferase are available from Xactagen: a secreted, highly stable enzyme (sgl) and a destabilized, membrane-anchored, and extracellularly displayed enzyme (mgl). Secreted enzyme can be sampled multiple times from supernatants while preserving cell integrity for downstream applications. The moderate half life of membrane-anchored enzyme is well suited for reducing lingering reporter activity that can mask effects of stimulators and inhibitors. Figure 1. Photo-oxidation of coelenterazine by Gaussia Luciferase. + O 2 + CO 2 + Light (hv = 475 nm) Gaussia luciferase can be ectopically expressed in mammalian cells using reporter plasmids available from Xactagen. Promoter / enhancer cloning vectors, pre-cloned promoters / enhancers in Xactagen vectors, and custom cloning of promoters / enhancers are available. Alternatively, Xactagen produces endogenous gene expression reporter cells (catalog and custom-made) wherein Gaussia luciferase is introduced into the natural gene of a mammalian cell so that gene regulation accurately reflects natural promoters, enhancers, suppressors, local chromosomal modifications such as DNA methylaiton, and mirnas. Reporter assays using Xactagen vectors, cells and Gaussia luciferase assay kits provide for ease of use, high signal to noise, and high reproducibility as required for quantitative analysis of the biological impact of extracellular signals, RNA interference reagents, protein over-expression, antibody treatment, peptide treatment, or small molecule drug treatment. They are well suited for assessing effects of single agents or for synergistic or antagonistic gene and drug interactions, either in cell culture or in animal models. GLum.1 Advantages Simple assays are performed at room temperature (bench top or automated). Higher Flash or Glow activities compared to competitive Gaussia luciferase reagents Flash or Glow with one reagent: no need to purchase alternative kits or add stabilizers Up to 1,000-fold higher activity than Renilla or firefly luciferase Assay when convenient feature: Following completion of an experiment, Gaussia luciferase may be refrigerated or frozen and assayed for activity when convenient ATP independent enzyme activity: monitors effects of stimulators and inhibitors on gene regulation, and not cellular metabolism Applicable for transient or stable expression vectors / cells Reliable linear results spanning 7 orders of magnitude. Weak promoters and strong promoters are all within the sensitivity range 3

4 Protocols: I. Secreted Gaussia Luciferase (sgl.1): A PREPARATION OF CELL CULTURES 1. Adherent and Nonadherent Cells: Adherent and nonadherent cells may be cultured in normal tissue culture vessels using standard growth media. See application note: linearity of assay. B. PREPARE ASSAY REAGENTS AND LUMINOMETER 1. Pre-warm GLum.1 Assay Buffer to room temperature 2. Prepare GLum.1 Assay Solution by adding GLum.1 Substrate to GLum.1 Assay Buffer. It is important to keep GLum.1 Assay Solution in the dark. Calculations are as follows: Reagent 96-Well Plates Volumes per Assay* 384-Well Plates Volumes per 100 Assays* Coelenterazine Substrate Solution 50 ul 40 ul GLum.1 Assay Buffer 5 ml 4 ml * Be sure to prepare additional GLum.1 Assay Solution for priming injectors as recommended by the manufacturer. C. PERFORM FLASH OR GLOW ASSAYS OPTION 1: FLASH ASSAYS STEP 1: Dispense 5 to 20 ul of sample into wells of a 96-well or 384-well white (opaque) plate. STEP 2: Prime the injector with GLum.1 Assay Solution per manufacturer s recommendations. STEP 3: Dispense (inject) GLum.1 Assay Solution into wells and measure luminescence Luminometer settings are as follows (e.g. for Molecular Devices, SpectraMax L): a. wavelength: ~475 nm b. Integration time: 5 seconds. c. Delay before beginning read: 0.1 seconds. d. Injection speed: 230 ul per second. e. Injection volume: 50 ul (96-well plates) or 40uL (384 well plates). f. Dark adapt: 1 minute. OPTION 2: GLOW ASSAYS. STEP 1: Dispense 5 to 20 ul of sample into wells of a 96-well or 384-well white (opaque) plate. STEP 2: Dispense 50uL GLum.1 Assay Solution into wells. Incubate 5 minutes at room temperature and read luminescence (1-5 second integration, ~ 475 nm) 4

5 II. Membrane-Anchored Gaussia Luciferase (mgl.1): A PREPARATION OF CELL CULTURES 1. Adherent Cells: Adherent cells should be plated into white (opaque) cell culture plates using standard growth media. See application note: linearity of assay. 2. Nonadherent Cells: Nonadherent cells can be cultured in normal tissue culture vessels. B. PREPARE ASSAY REAGENTS AND LUMINOMETER 1. Pre-warm GLum.1 Assay Buffer to room temperature You will need 100uL per assay in 96-well format or 80uL per assay in 384-well format 2. Prepare GLum.1 Assay Solution by adding GLum.1 Substrate to pre-warmed, GLum.1 Assay Buffer (keep in the dark). Calculations are as follows: Reagent Coelenterazine Substrate Solution GLum.1 Assay Buffer 96-Well Plates Volumes per 100 Assays* 384-Well Plates Volumes per 100 Assays* 50 ul 40 ul 5 ml 4 ml * Be sure to prepare additional GLum.1 Assay Solution for priming injectors as recommended by the manufacturer. C. PERFORM FLASH OR GLOW ASSAYS OPTION 1: FLASH ASSAYS STEP 1: Prepare assay plates for Gaussia luciferase assay a. Adherent cells: Aspirate media from cells and replace with pre-warmed, GLum.1 Assay Buffer (50 ul per well for 96-well format; 40 ul per well for 384-well format). b. Non-adherent cells: Resuspend cells and pipet 5 to 50 ul cells for 96-well format 5 to 20 ul cells for 384-well format STEP 2: Prime the injector with GLum.1 Assay Solution per manufacturer s recommendations. 5

6 STEP 3: Dispense (inject) GLum.1 Assay Solution into wells and measure luminescence Luminometer settings are as follows (e.g. for Molecular Devices, SpectraMax L): a. wavelength: ~475 nm b. Integration time: 5 seconds. c. Delay before beginning read: 0.1 seconds. d. Injection speed: 230 ul per second. e. Injection volume: 50 ul (96-well plates) or 40uL (384 well plates). f. Dark adapt: 1 minute. OPTION 2: GLOW ASSAYS. STEP 1: Prepare assay plates for Gaussia luciferase assay a. Adherent cells: Aspirate media from cells and replace with pre-warmed, GLum.1 Assay Buffer (50 ul per well for 96-well format; 40 ul per well for 384-well format). b. Non-adherent cells: Resuspend cells and pipet 5 to 50 ul cells for 96-well format 5 to 20 ul cells for 384-well format STEP 2: Dispense 50uL GLum.1 Assay Solution into wells. Incubate 5 minutes at room temperature and read luminescence (1-5 second integration, ~ 475 nm) 6

7 Application Notes: 1. Freezing Samples: Secreted Gaussia luciferase (sgluc) is a stable enzyme. It may be stored refrigerated for several days or frozen at -20 o C for several weeks without loss of enzymatic activity. Thaw frozen samples and equilibrate to room temperature before proceeding with the Gaussia Luciferase Assay. 2. GLum.1 Assay Solution: GLum.1 Assay Solution should be protected from light at all times and should be used within 24 hours of preparation. Unused Assay Solution may be frozen at -20 o C. Thaw frozen Assay Solution and equilibrate to room temperature before proceeding with the Gaussia luciferase Assay. 3. Background Determination: The Gaussia Luciferase Assay should be performed using growth media (secreted Gaussia luciferase) or Gaussia-luciferase-free cells (destabilized, membrane-anchored Gaussia luciferase). The presence of serum in samples adds to background luminescence. For most applications, serum contribution is negligible compared to signal. However, for very weak promoters, higher signal to noise may be attained by keeping serum (media with serum) to a minimum. 4. Linearity of Assay: The linear range of luminometers varies by manufacturer making it necessary to establish the linear range of the assay. a. For secreted Gaussia luciferase (sgl), this is done by serially diluting samples, measuring Gaussia luciferase activity, and plotting relative luminescence units (RLUs) per volume of sample. If activity exceeds the instrument range, samples should be diluted in PBS or serum-free growth media. b. For membrane-anchored Gaussia luciferase (mgl), this is done by plating serially diluted cells into white (opaque) tissue culture treated culture plates, measuring Gaussia luciferase activity after at least a 16 hour incubation time, and plotting relative luminescence units (RLUs) per cell. Use cell densities within the linear range for the assay (Figure 2). 7

8 Membrane-Tethered, Extracellular Gaussia Luciferase (Activity as a Function of Cell Density) Relative Luminescence Units (RLUs) ,000 10,000 15,000 20,000 25,000 30,000 Cells Plated per Well Figure 2. The Gaussia luciferase reporter gene was introduced into endogenous genes in HCT116 human colon cancer cells. A cloned reporter cell was serially diluted and plated into wells of a 96-well, white wall and bottom cell culture plate. Gaussia Luciferase Assay was performed 18 hours later to determine the linear range for the assay. Conclusion: The assay was linear from as few as 500 cells (lowest amount plated) to 20,000 cells per well. Background luminescence was 1,388 +/- 152 RLUs. References: 1. Szent-Gyorgyi, C., et al. (1999). Cloning and characterization of new bioluminescent proteins. part of the SPIE Conference on Molecular Imaging: Reporters, Dyes, Markers, and Instrumentation. San Jose, CA. Proc. SPIE. 3600:4 2. Tannous, B.A. et al. (2004). Codon-optimized Gaussia Luciferase cdna for Mammalian Gene Expression in Culture and in Vivo. Molecular Therapy. 11: Verhaegen, M. and Christopoulos, T.K. (2002) Recombinant Gaussia luciferase. Overexpression, purification, and analytical application of a bioluminescent reporter for DNA hybridization. Anal. Chem. 74,