Building an Enzyme Profile with Tecan’s D300e Digital Dispenser

The Tecan  D300e Digital Dispenser provides breakthrough capabilities for life sciences dispensing workflows. The dispenser removes the need for serial dilutions, and allows researchers to effortlessly miniaturize experiments at the bench.

Thanks to HP’s inkjet technology, picoliter to microliter volumes of tiny molecules and biomolecules in surfactant-containing aqueous solutions or DMSO are dispensed directly into most standard microplate formats.

Users can select from several aqueous-based fluid classes, other than DMSO, through the ‘fluid classes’ feature in the D300e software. The software eliminates the necessity to perform complex fluid calibration adjustments which are needed with other technologies, and optimizes the user experience.

Tecan D300e Digital Dispenser

Figure 1. Tecan D300e Digital Dispenser

Through the Tecan  D300e software, researchers can easily create both simple and complex protocols, involving single or multiple destination microplates and one or more fluids.

The user interface has a familiar look and feel, consists of one main screen, and many functions that can be accessed by contextual right-click menus. The user can easily add an aqueous fluid to a protocol and select the correct fluid class. Table 1 shows the fluid classes which are currently enabled for use.

Table 1. Fluid classes available for dispensing with the Tecan D300e

Fluid class

Surfactant

Surfactant conc.

Glycerol

Biomolecules

Small molecules

DMSO (70-100%)

N/A

N/A

N/A

<300 kDa, <3 mg/ml

<10 mM

Aq + Brij™ 35

Brij 35

0.1%

<5%

<300 kDa, <3 mg/ml

<10 mM

Aq + Brij 35 + gly

Brij 35

0.1%

5-20%

<300 kDa, <3 mg/ml

<10 mM

Aq + Triton™ X-100

Triton X-100

0.1%

<5%

<300 kDa, <3 mg/ml

<10 mM

Aq + Triton X-100 + gly

Triton X-100

0.1%

5-20%

<300 kDa, <3 mg/ml

<10 mM

Aq + Tween® 20

Tween 20

0.3%

<5%

<300 kDa, <3 mg/ml

<10 mM

Aq + Tween 20 + gly

Tween 20

0.3%

5-20%

<300 kDa, <3 mg/ml

<10 mM

Building a fluid list

The Fluid List provides details on to-be-tested fluids, including the class of the fluid, which can be chosen from a drop-down menu as shown in Figure 2.

After a fluid class is chosen, the software automatically considers the difference in dispensing volume increments, cup overfill volumes, and minimum dispense volumes. Additionally, when an aqueous fluid class is chosen, the protocol will be restricted to one 1,536-well or four lower-density microplates, thus reducing the influence of evaporation on the assay results.

Example Fluid List in Tecan D300e software

Figure 2. Example Fluid List in Tecan D300e software

Normalization of aqueous fluids following dispensing

When surfactant-containing aqueous fluids are dispensed, it may be convenient to have a constant surfactant concentration per well for some assays. Thanks to the software’s Normalization feature, the DMSO or surfactant content in the assay wells can be easily normalized, according to the highest volume well, a user-specified volume, or a user-specified percentage.

When the normalization fluid is dispensed using a T8+ Dispensehead Cassette, the dispense volume ranges from 1 nl to 20 μl (maximum 10 μl per well), whereas the D4+ Dispensehead Cassette’s volume ranges from 1 nl to 200 μl (maximum 10 μl per well).

Normalization fluids must not contain any small molecule compounds or biomolecules. In protocols with multiple types of fluids, individual wells can be sequentially normalized with more than one normalization fluid.

In assays that will be read spectrophotometrically directly from the assay plate (with no staining or washing steps), it is recommended that the surfactant level be normalized, so that the results are not affected by surfactant-driven meniscus differences (Figure 3).

Effect of Tween 20 normalization on fluorescein fluorescence intensity across a range of wells containing the same nominal fluorescein concentration

Figure 3. Effect of Tween 20 normalization on fluorescein fluorescence intensity across a range of wells containing the same nominal fluorescein concentration

Enzyme profile function

By using the new Enzyme Profile function, users can conveniently program complex enzyme studies – Km determination, enzyme rate determination, and Lineweaver-Burk studies. For this purpose, up to two enzymes, up to two substrates or co-factors, and any number of inhibitors can be used.

Based on the aim of the experiment, different components can be titrated or held constant. Figures 4 and 5 present data produced from a histone deacetylase (HDAC) assay. Some of the Enzyme Profile function steps that are used to build the protocols are shown in Examples 1 and 2 (not all steps are shown).

Kinetic data from HDAC activity assay using a fixed concentration of substrate

Figure 4. Kinetic data from HDAC activity assay using a fixed concentration of substrate

Michaelis-Menten data from HDAC activity assay

Figure 5. Michaelis-Menten data from HDAC activity assay

The activity of an enzyme could be tested by titrating the enzyme in combination with a fixed substrate concentration. A) Select one enzyme and one substrate. B) Select enzyme for titration (substrate will be held constant). C) Set enzyme titration details and substrate concentration. D) Plate layout is automatically generated

The activity of an enzyme could be tested by titrating the enzyme in combination with a fixed substrate concentration. A) Select one enzyme and one substrate. B) Select enzyme for titration (substrate will be held constant). C) Set enzyme titration details and substrate concentration. D) Plate layout is automatically generated

The activity of an enzyme could be tested by titrating the enzyme in combination with a fixed substrate concentration. A) Select one enzyme and one substrate. B) Select enzyme for titration (substrate will be held constant). C) Set enzyme titration details and substrate concentration. D) Plate layout is automatically generated

The activity of an enzyme could be tested by titrating the enzyme in combination with a fixed substrate concentration. A) Select one enzyme and one substrate. B) Select enzyme for titration (substrate will be held constant). C) Set enzyme titration details and substrate concentration. D) Plate layout is automatically generated

Example 1. The activity of an enzyme could be tested by titrating the enzyme in combination with a fixed substrate concentration. A) Select one enzyme and one substrate. B) Select enzyme for titration (substrate will be held constant). C) Set enzyme titration details and substrate concentration. D) Plate layout is automatically generated

Km determination for substrate using fixed enzyme concentration. A) Select substrate for titration (enzyme will be held constant). B) Set substrate titration details and enzyme concentration. C) Plate layout is automatically generated.

Km determination for substrate using fixed enzyme concentration. A) Select substrate for titration (enzyme will be held constant). B) Set substrate titration details and enzyme concentration. C) Plate layout is automatically generated.

Km determination for substrate using fixed enzyme concentration. A) Select substrate for titration (enzyme will be held constant). B) Set substrate titration details and enzyme concentration. C) Plate layout is automatically generated.

Example 2. Km determination for substrate using fixed enzyme concentration. A) Select substrate for titration (enzyme will be held constant). B) Set substrate titration details and enzyme concentration. C) Plate layout is automatically generated.

Conclusion

The Tecan D300e Digital Dispenser offers the power of HP’s inkjet technology to life sciences research, and the software enables generating even the most complex plate layouts in an easy manner. The addition of aqueous dispensing feature means that the Tecan D300e Digital Dispenser can now complete even more work, including complex enzyme profiling studies.

About Tecan

Tecan is a leading global provider of automated laboratory instruments and solutions. Their systems and components help people working in clinical diagnostics, basic and translational research and drug discovery bring their science to life.

In particular, they develop, produce, market and support automated workflow solutions that empower laboratories to achieve more. Their Cavro branded instrument components are chosen by leading instrumentation suppliers across multiple disciplines.

They work side by side with a range of clients, including diagnostic laboratories, pharmaceutical and biotechnology companies and university research centers. Their expertise extends to developing and manufacturing OEM instruments and components, marketed by their partner companies. Whatever the project – large or small, simple or complex – helping their clients to achieve their goals comes first.

They hold a leading position in all the sectors they work in and have changed the way things are done in research and development labs around the world. In diagnostics, for instance, they have raised the bar when it comes to the reproducibility and throughput of testing.

In under four decades Tecan has grown from a Swiss family business to a brand that is well established on the global stage of life sciences. From pioneering days on a farm to the leading role our business assumes today – empowering research, diagnostics and many applied markets around the world


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Last updated: Apr 1, 2019 at 5:19 AM

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