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  LigandTracer

Kinetic information

You get information about the rates of the interaction. For example, the off-rate often correlates with the duration of effect of a ligand.

 

Live cells

Measuring interactions on cells takes you one step closer to in vivo. Targets can be naturally expressed in the cell membrane, at physiological levels, in the presence of cellular molecules and processes that may affect the binding.

 

Follow the interaction as it occurs

You can confirm a binding at an early stage and can adapt the measurement based on signal increase and observed rates. There is no requirement to reach equilibrium and the high data frequency reduces the risk of blind spots.

 

Saves time

Few preparatory steps are needed and since the instrument is semi-automated you can do other things while the measurement proceeds.

 

No or a few washes required

Washing is a cumbersome step in most manual methods. Washes are also critical when having fast off-rates, since much of the ligand risks being washed off before detection with manual methods.

 

Simple to operate

Our intuitive control software and simple technology ensures that minimal training is required to operate the instrument.

 

Much support material

For a deeper understanding on the hows and whys of LigandTracer we provide a learning software and offer courses.

  • One of the binding partners (= target) must be immobilized on a Petri or glass dish; this can e.g. be receptors of adherent cells, anchored immune cells, or tissue sections.
  • The binding partner in solution (= ligand) must be labeled with either fluorescence or radioactivity. The instrument models vary on which labels they can detect. 
  • Sufficient amount of targets. For a confluent cell layer we recommend at least 105 target receptors/cell.

Regular cell culture media is recommended when measuring with live cells. Cells can be seeded in standard “10 cm” Petri dishes (Ø: 87-89 mm and h: 15-18 mm without the lid) – vendor independent. Optionally, to increase throughput, LigandTracer MultiDish 2×2 can be purchased for 2nd generation LigandTracer Green instruments.

There are no maintenance cost for LigandTracer. However, we do offer a service contract for LigandTracer Green (2nd generation) that includes an instrument qualification, certificate, one year extended warranty and a possible detector update if the signals are deemed too low.

We recommend starting with room temperature measurements, to learn about the interaction with less interference from cellular processes. In our experience many cell lines can handle being at room temperature or even in cold rooms for many hours.

For measurements at 37 °C we recommend using an incubator and not the heating unit of the instrument, in particular for LigandTracer Green.

It is not possible to narrow the detection energy range to such an extent that the different radionuclides can be distinguished. Please refer to the product specification of each instrument model for information about detectable nuclides.

No, all software are included in the price and pre-installed upon delivery.

In case the application is new, we offer a 4-week on-site trial of our LigandTracer system with closed wallets; we ship the system to you and you ship it back to us. Our support team is available by email during the trial to discuss your assay set-up, optimization and results. We can set up phone or online meetings as well to help you plan and carry out your trial.

Alternatively, you can lease a LigandTracer for a 3-month period for your project.

  Support

Our interactive training software LigandTracer Learning is a good starting point. It describes not only how to evaluate interactions with LigandTracer, but also why we do it that way.

Additionally, we offer courses in LigandTracer analysis approximately once a year. In the course we measure interactions in real-time with LigandTracer and analyze the data with TraceDrawer. We also offer on-site training, for example coupled to an instrument purchase. Please get in touch with our team for more information.

If you have a starting guess of the affinity: Choose a concentration close to or slightly above KD.

If not: Start with 1 nM. If you do not see a signal during the first 30 min, increase the concentration three times. Repeat until you observe a binding.

Baseline

Measurement of the signal before ligand has been added.

 

Association phases

Measurement of the signal increase after ligand has been added. At least two association phases at different ligand concentrations are recommended. The aim of additional incubation phases is to obtain more information about the interaction, which in turn increases the accuracy of the evaluation of affinity and kinetics.

 

Dissociation phase

Measurement of the signal decrease after ligand has been removed, to follow the dissociation rate.

Evaluate your real-time binding data with our software TraceDrawer. The handbook and step-by-step examples can be found in the Help menu of TraceDrawer.

We recommend 3 ml for regular Petri dishes and 1.8 ml in each compartment (=each half) of a LigandTracer MultiDish 2×2.

Ensure that you see a clear signal increase for each association phase. Continue until you see curvature, i.e. when the signal increase is no longer linear and starts to have less slope. Note that no signal increase is expected if all targets were already saturated during incubation with the previous concentration.

If possible, continue the dissociation measurement until you observe a signal decrease of at least 10 %. For very stable interactions, such as with some therapeutic antibodies, this may take many hours.

Place the instrument in the incubator at least 1 h (LigandTracer Yellow, Grey or White) or 6 h (LigandTracer Green – e.g. the evening before) prior to measurement to ensure proper temperature equilibration.

It is important to remove LigandTracer from the incubator within 24 h (LigandTracer Yellow, Grey and White) or 24 h (LigandTracer Green) and allow it to stabilize at room temperature for 12-24 h before the next incubator measurement.

  • Study binding to a control, e.g. a negative cell line with minimal target expression. Control cells and target cells can be included in the same measurement, at different positions of the dish.
  • For interactions that are nicely fitting to a OneToOne model, you basically have enough evidence as the OneToOne model assumes specificity to a target.
  • Pre-incubate the targets with non-labeled ligand. Add labeled ligand and study if binding can occur. If there is no clear self-competition the binding may be non-specific.

 

See our application Specificity and competition for more information.

A: Competition measurement

Measure binding of a labeled ligand in the present of a non-labeled ligand. The non-labeled ligand could either be pre-incubated or added after some labeled ligand has been bound. If the binding of the labeled ligand is not affected there is likely no competition.

 

B: Proximity measurement*

Start by performing a competition measurement (A) to ensure that there is no competition. Label one of the ligands with a quencher and see if the signal from the other ligand (fluorescently labeled) is affected when adding the quencher ligand. A signal disruption corresponds to close proximity, assuming that there is no competition observed in (A), suggesting that both ligands can bind the target simultaneously.

*Only for LigandTracer Green

See our application Specificity and competition for more information.

We recommend using adsorbed antibodies targeting surface proteins of the bacteria. Read the article Detecting ligand interactions in real time on living bacterial cells for more information.

It is important that the cells stay attached during the experiment to keep the number of targets constant and achieve reliable interaction data. Coatings such as polydopamine can improve the attachment for adherent and semi-adherent cells during LigandTracer measurements. Please refer to our protocols Coat with polydopamine to enhance cell adhesion and Seeding cells in polydopamine coated dishes.

There are a large number of pre-labeled ligands and labeling kits on the market. We also have several labeling protocols in Resources.

Absence of a signal increase may for example be due to:

  • Too low concentration of ligand: A concentration equal to KD results in 50 % of the targets being bound at equilibrium. With 0.1×KD it is only 9 %. Try to increase the concentration.
  • More time is needed: Some interactions are slow and are barely detectable the first 30 min. In those cases you must incubate a bit longer to obtain a signal increase.
  • Too few targets: We recommend having a total of 1011 This is typically achieved with a medium to high expression level and cell confluency approaching 100 %.
  • Labeling gone wrong: Troubles during labeling, resulting in a very low labeling degree, can make it difficult to detect a signal. The label itself may also affect the binding behavior of the ligand.
  • Non-specific binding to the reference area: If there is much non-specific binding to the reference position any binding to the target area may be lost.

 

Please contact us if you have troubles with your measurements and we will try to solve it together with you.

Yes, before you add/remove media or ligand you can rotate the cell dish holder by hand to avoid accidentally touching the cells with the pipette tip or exposing cells to turbulent flow and/or unequilibrated ligand concentrations during ligand addition. The cell dish holder is automatically turned to its home position when resuming the experiment.

Make sure to never move/remove the dish from the cell dish holder during the experiment.

Yes, LigandTracer measurements are done without the dish lid.

Our Experiment planner in LigandTracer Control (Tools menu) will help you calculate the volumes needed to obtain the concentration series of your choice.

The heating/cooling unit is convenient for short measurements in LigandTracer Yellow, Grey and White. However, since it tends to increase medium evaporation it is not recommended for LigandTracer Green, which is sensitive of condensation on the lenses, or long measurements in Yellow, Grey or White.

Our detectors are very sensitive, unfortunately, also to electromagnetic disturbances. For example, answering a phone call when standing close to the LigandTracer might result in a signal spike. Surges from electronic equipment in vicinity or coupled to the same power supply circuit can also cause signal disturbances. Changing the power outlet group or adding a power stabilization & protection UPS (for example this) might solve the problem.

For LigandTracer Green the disturbance may also be due to an incorrect positioning of the detector. Push the detector down, towards the detector holder, to ensure that there is perfect connection between detector and instrument.

We have seen such signal oscillations when ligands aggregate to small balls that “roll” over the surface of a Petri dish. Improving the solubility of the labeled ligand will solve the problem.

No, TraceDrawer is not compatible with Mac OS. For Mac users we recommend running TraceDrawer in a Windows emulator, such as Parallels. TraceDrawer is compatible with Windows 7 or Windows 10.

  Orders, terms and delivery

Please contact us to request a quotation.

Delivery time is up to 8 weeks after order.

The off-site support included in an instrument purchase is specified in the quotation, but is often 10 h.

Standard warranty is 1 year from date of delivery, please inquire about options for longer warranty.

The delivery terms are according to DDU (INCOTERMS 2000).

The course price includes participation at lectures/exercises, printed handouts, reagents in the wet-lab and all meals except breakfast.

Flight and accommodation are not included.

Payments are typically done as wire transfers, but we can also accept PayPal payments.

Can’t find the answer to your question? Contact us for assistance.