heliX®
Experimental Considerations
With a simple dye scouting procedure you can easily identify the most suitable dye for your application in only 2 hours. You use the dye scouting kit and perform the usual steps of ligand preparation, functionalization and a standard kinetic assay with the different dyes in our dye scouting kit. Then you can choose the dye that yielded the highest change in fluorescence for your ligand-analyte pair.
The adapter strands are available with three different red and three different green fluorescent probes. The dyes differ in chemical properties to optimize the signal for your specific application. In general, we recommend to use the dye Ra, since it offers highest stability even throughout long measurement times. To screen for the most sensitive fluorophore for your application you can perform a dye scouting.
The heliX+ chip has two sensor spots and each sensor spot allows for two-color detection, enabling you to simultaneously detect four signals by four single-photon detectors. You can either multiplex four interactions or use one spot for real-time referencing. Please note that the heliX in contrast to the heliX+ only allows for single-color detection.
Yes! You can either functionalize the surface with two different ligands and differentiate between affinity and avidity. Or you can use the specific DNA Y-structure to examine bispecific analytes, ternary complex formation or protein dimerization.
Yes, with a heliX+ you can multiplex up to four interactions due to four single-photon detectors. Two colors can be detected on each of the two sensor spots. And if you want to scale up your throughput, you can combine several heliX+ instruments in one heliOS network.
The heliX+ instruments were designed to fit your specific needs. You can easily scale up your throughput by combining as many heliX+ modules as you require. This modular design offers high versatility – you can combine heliX units in one heliOS network, operate them simultaneously or in parallel. Build your custom high throughput network or ask us for our special offer heliX+ packages.
We know your samples are precious. So we optimized the switchSENSE® technology for minimum sample consumption. You will need approximately 5 pmol ligand per experimental run. Of course, this highly depends on your exact experimental conditions. You can always contact us if you need help with your specific experimental set-up.
Yes, the heliX+ instrument has three different buffer lines. One is used for the maintenance buffer, facilitating standard protocols like functionalization. The other two can be equipped with your individual measurement buffers. The heliOS software has a smart priming feature that remembers the buffer last used for each buffer line and automatically includes a priming step in case the buffer has been changed.
For kinetic experiments, choosing the appropriate flow rate can be a crucial factor for determining the correct affinity. Insufficient flow rates can potentially lead to measurement artifacts. During the association phase, a too slow flow rate can be the cause for mass transport limitation effects. During the dissociation phase an inadequate flow rate can result in re-binding effects. Both these effects should be avoided to obtain high quality kinetic data. Thus, the general rule is to work at high flow rates (> 100 µl/min) whenever the experimental circumstances allow.
It is advisable to use a detergent-free running buffer (e.g. PE40 without Tween-20). Liposomes can for example be immobilized on the chip surface using a cholesterol-ligand, i.e. cholesterol modified DNA nanolever (cholesterol intercalates into lipid bilayer). Use low flow rates for association and dissociation (e.g. < 50 µl/min) to prevent destabilization of membranes.
Yes, you can. And there’s more to it than that. You can even differentiate between the association of DNA/RNA binding enzymes and their catalytic activity and independently examine the effects of inhibitors on these processes.
You can separately adjust the chip tray temperature (i.e. the temperature of your measurement) and the sample tray temperature. This allows you for example to keep your samples cold, but measure at higher temperatures. The chip tray temperature can be adjusted to 15 °C – 40 °C. The temperature range for the sample tray is 4 °C – 40 °C.
Generally, switchSENSE® can tolerate measurements in a variety of complex matrices, including cell lysates and high serum concentrations up to 80 %. Nevertheless, measurements in complex media usually require a certain degree of assay development. In most cases, this is owed to the occurrence of unspecific adsorption to surfaces of the sensor system. For successful experiments, please consider the following aspects:
- Always filter samples or remove solid components by centrifugation prior to injection.
- The addition of non-specific competitor substances (fragmented salmon sperm DNA, polydIdC) is usually required to reduce background affinity.
- When using salmon sperm DNA or similar reagents, make sure to use a well buffered solution to avoid acidification.
- Measurements in static mode are usually more robust when complex media are tested.
- The activity of nucleases that are potentially present in the test solution, is often reduced by EDTA.
- Always perform a reference injection on DNA nanolevers without ligand.
- As complex media usually differ significantly in composition, the ideal dilution of the original sample should be optimized.