How active are enzymes? This is an often addressed question in biochemical laboratories. As enzymes are the catalyzers of most chemical reactions taking place in an organism, they and their regulators are often characterized in detail.
Enzymatic assays typically employ a substrate that is converted to a chromophore, ﬂuorophore or a luciferase substrate in course of the enzymatic reaction. This means in turn that the signal increases with increasing reaction time until all the substrate is converted by the enzyme. As it is hard to foresee at which range of signal intensity an enzymatic assay ends their detection is challenging and is often associated with various rounds of ﬁnding the right ampliﬁ cation for ﬂuorescent and luminescent signals. A virtually unlimited dynamic range as found with the enhanced dynamic range (EDR) feature drastically simpliﬁes ﬂuorescent and luminescent enzyme assays and saves time to set up the measurement.
The enzyme β-galactosidase (β-gal) catalyzes the hydrolysis of β-galactosides by breaking up glycosidic bonds. It is used in biological research to report on cellular senescence which is indicated by senescence-associated β-gal. A second and very popular use of β-gal is as a reporter gene: a DNA construct contains the genetic information for β-gal and a promoter that regulates the expression of the enzyme. Mostly, β-gal serves as control gene. This means it is inserted into the cells of interest during transient transfection along with a reporter gene of interest. In this case β-gal reports on the transfection efﬁciency. A high transfection efﬁciency results in high enzyme production; low transfection efﬁciency results in low enzyme expression. The amount of expressed β-gal can be tested with a non-ﬂuorescent synthetic β-gal substrate: Fluorescein di (β-D-galactopyranoside) (FDG). It is cleaved by the enzyme to galactose and the ﬂuorescent molecule ﬂuorescein (FITC) (Fig. 1). Here, we measured various enzyme concentrations on the CLARIOstar Plus with and without use of the novel EDR feature.
The enhanced dynamic range (EDR) refers to a technology that measures very dim and very intense signals in one plate measurement. This way, a dynamic range of 8 decades can be covered. In contrast, traditional measurements require an ampliﬁcation of the signal that is typically set by the gain. This limits the range in which signals can be detected (Fig. 2).
- Black 96-well plate with ﬂat bottom (Greiner bio-one # 655076)
- CLARIOstar Plus
- β-Galactosidase from Escherichia coli (G6008-1KU, Sigma-Aldrich / Merck)
- Fluorescein Di-β-D-Galactopyranoside (F1179, Invitrogen / Thermo Fisher Scientiﬁc)
The assay buffer was prepared by weighing 0.8370 g KH2PO4, 0.6855 g Na2HPO4·2H2O, 0.0625 TCEP and 0.0203 g MgCl2·6H2O, dissolving in distilled water and adjusting the volume to 50 ml (ﬁll up to 50 ml).
The β-Galactosidase was dissolved in 1 ml of distilled water (enzyme stock solution) and the substrate ﬂuorescein Di-β-D-Galactopyranoside was dissolved in 10 ml of water (0.5 mg/ml substrate buffer). Standards of 0.5 U/ml; 0.25 U/ml; 0.1 U/ml; 0.05 U/ml; 0.025 U/ml; 0.01 U/ml were prepared by preparing double of the end concentration in distilled water and subsequently diluting it 1:2 in substrate buffer. The substrate concentration was kept constant at 0.25 mg/ml. Total reaction volume was 200 µl and four replicates were measured. Fluorescence intensity measurements were performed by the CLARIOstar Plus multi-mode microplate reader with the settings indicated below.
|Fluorescence intensity, Plate mode kinetic
|EDR or gain as indicated
Number of flashes
Number of cycles
Results & Discussion
In order to test the suitability of the CLARIOstar Plus for measuring β-gal reporter assays, different enzyme concentrations were incubated with the synthetic substrate FDG and its conversion was monitored by measuring FITC ﬂuorescence every 10 minutes. Traditionally, a signal range needed to be chosen up-front in order to detect such a kinetic measurement. For demonstration purpose, we selected a low gain (800) which translates to low signal ampliﬁcation and should prevent excessive ampliﬁcation of the increasing ﬂuorescent signal.
However, detection in this wisely preselected range resulted in overﬂow measurements for all of the enzyme concentrations as the signal increased further than expected (Fig. 3). The lowest enzyme concentration showed an increase in ﬂuorescence until 1 h 10 min after reaction start (orange line Fig. 3), but ﬂattened out the following timepoints as the signal exceeded the preselected intensity range. A quantiﬁcation of the enzyme is not possible with the acquired data.
Measuring the same plate with the EDR feature does not require any preselection. It results in the resolution of each enzyme concentration, from the highest (0.5 U/ml) down to the lowest (0.01 U/ml) (Fig. 4). Thus, the EDR feature prevents the need to prepare and measure a second time. Besides that it reduces the time to run a working enzyme assay as well as concerns when starting the measurement. The data further provided a linear correlation between maximum conversion speed and enzyme concentration (R² >0,99) allowing quantiﬁcation of the enzyme up to 0.1 U/ml (data not shown).
Here, we demonstrated the detection of a β-gal enzyme assay that is primarily used for reporter assays. With the help of the EDR technology of the CLARIOstar Plus β-gal mediated production of ﬂuorescein was conveniently monitored. It allowed the detection of huge signal differences covering a range from 660 RFU up to 3.44*108 RFU. Translated to a reporter assay, this means very high and very low expressions can be measured at the very same plate. The tool simpliﬁes the set-up of enzymatic assays as it does not require time to think about the right gain settings or repeat the assay until optimal settings are found.