One-Step Luciferase Assay System

Catalog Number: LAS-1 10ml; LAS-2 100ml

Description

The One-Step Luciferase Assay System is designed to be used for high-throughput, sensitive quantitation of firefly luciferase activity in mammalian cell culture. The reagent consists of two components, a Luciferase Reagent Buffer (Component A) and Luciferase Reagent Substrate (Component B). Component A and Component B are combined to form a working solution that contains all the necessary components for cell lysis and luciferase quantitation. This assay system has several features:

•	Sensitive – highly sensitive detection of firefly luciferase activity.
•	Stable – the signal output is stable for more than two hours, providing flexibility with regard to incubation time
•	Convenient – simple one-step, homogeneous protocol.
•	High-throughput – one-step homogeneous protocol minimizes handling steps to support high-throughput screening applications
•	Compatibility – works well with a variety of common media containing 0-10% serum and phenol red.
•	Instrumentation – does not require a luminometer with injectors.

Application
•	Monitor firefly (Photinus pyralis) luciferase activity in cultured mammalian cells.
•	High-throughput screening using luciferase reporter cell lines

Components

Component	Amount(LAS-1)	Amount(LAS-2)	Storage	
Luciferase Reagent Buffer
(Component A)	10ml	2x50ml	-20°C	
Luciferase Reagent
Substrate, 100x
(Component B)	100 µl	2x500ul	-20°C
Protect from light	
Each system contains sufficient reagents to perform 1,000 assays of 100 μl each in 96-well plate.

Stability
At least 6 months when stored as directed. Upon first thaw, store in aliquots at -20°C.   The reagent may be subjected to several freeze/thaw cycles with no effect on functionality, but it is recommended that freeze/thaw cycles be avoided whenever possible.

Background
Luciferase is the general term given to a class of oxidative enzymes that catalyze reactions that give off light, a process known as bioluminescence (Fig. 1). In biology, researchers can take advantage of this reaction and use it as a readout for various biological processes. This has perhaps been exploited most in luciferase reporter cell lines where a promoter region from a gene of interest is placed immediately upstream of the coding sequence for luciferase. In this system, transcriptional activation of the gene of interest leads to a level of luciferase expression that is proportional to the level of gene activation.

The level of activation is then assessed by lysing the cells and adding the luciferase substrate, D-luciferin. Using the One-Step Luciferase Assay System from BPS Bioscience, this is accomplished in a single step as the lysis buffer has been formulated with D-luciferin. After a brief incubation period, bioluminescence is read on a luminometer.

Figure 1. Bioluminescent reaction catalyzed by luciferase. In the luciferase reaction, the cells are lysed with  luciferase  substrate  containing D-luciferin. Firefly luciferase derived from reporter cells, using ATP and Mg2+  as a co-substrate, derived from catalyzes  the  conversion  of  D- luciferin to oxyluciferin in a reaction that  gives  off  light.  The  amount  of light given off is proportional to the amount of luciferase present in the reaction  and  thus,  correlates  with gene   activation.   Luminescence   is read on a luminometer.

Important Product Information

•	The reagent has been validated in a 96-well format. Other formats will require scaling and optimization by the end- user.
•	Luciferase Reagent Buffer must be at ~ room temperature (20- 25°C) before use.

•	Luciferase  Assay  Reagent  should  be  added  to  cells  for  at  least  5  minutes  before measuring luminescence to allow complete cell lysis.
•	For maximal light intensity, measure samples within 1 hour of reagent addition.
•	Avoid exposing to excessive heat or light during incubation.
•	Different cell lines may exhibit variation in lysis ability and/or luminescence signal and may require slight optimization by the end-user.
•	Luminescence  signal  is  affected  by assay conditions.  Results should be  compared between samples measured using the same cell type and media/serum combinations.
•	To  analyze  multiple  plates,  include  a  common  control  sample  in  each  plate  and normalize the luminescence of each plate to the control contained in the same plate.
•	Background luminescence is a characteristic of luminometer performance, therefore, background luminescence must be subtracted from all readings for accuracy.

Materials Required but Not Supplied
•	Multiwell tissue culture plates that are compatible with luminometer being used
•	Mammalian cells that express firefly luciferase
•	Appropriate cell culture medium
•	Laboratory platform shaker
•	Luminometer

General Assay Procedure

1. Thaw Luciferase Reagent Buffer (Component A) by placing the reagent in a room temperature water bath. Equilibrate the buffer to room temperature and mix well before use.

2.  Calculate the amount of Luciferase Reagent needed for the experiment (Component A + Component B). Immediately prior to performing the experiment, prepare the luciferase assay working solution by diluting Luciferase Reagent Substrate (Component B) into Luciferase Reagent Buffer (Component A) at a 1:100 ratio and mix well. Avoid exposing to excessive light. Only use enough of each component for the experiment, remaining Component A and Component B should be stored separately at -20°C.

3.  Remove multi-well plate containing mammalian cells from incubator. Note: plates must be compatible with luminescence measurement with luminometer being used.

4.  Add equal volume of luciferase assay working solution (Component A + Component B) to the culture medium in each well. Example: 96-well plate with 100 µl of culture medium requires 100 µl of luciferase assay working solution per well.

5.  Gently rock the plates for ≥15 minutes at room temperature. Measure firefly luminescence using a luminometer.

The signal under these conditions will be stable for more than 2 hours at room temperature. For maximal light intensity, measure samples within 1 hour of reagent addition.