How the unrivaled Gator Plus+ plate cooling unlocks new assay possibilities

The Gator Plus+ runs BLI assays from 10°C to 40°C. Below are the applications unlocked by that range.

Keeping it cool

Biolayer interferometry (BLI) is typically valued for its speed, simplicity, and sensitivity. BLI assays typically run between 25°C and 37°C. The Gator Plus+ runs from 10°C to 40°C, a wider range than any previous BLI instrument. This expanded range enables experiments that weren’t possible before, like working with low-stability reagents, measuring kinetics values form fast-on/fast-off binders, and observing the thermodynamic and mechanistic characteristics of an interaction using variable temperatures.

1. Working with Low-Stability Reagents

Not every binding partner is stable at room temperature. Many therapeutic and diagnostic molecules (like RNA, peptides, nanobodies, and lipid nanoparticles) aren’t stable for hours of experimentation at 25-37°C. When these molecules degrade or aggregate, unusual or inconsistent experimental results often follow, and these inconsistencies can invalidate dozens if not hundreds of candidate experiments.

However, it’s not just the drug candidates with stability issues – many potential drug targets have remained untargeted due to their poor stability. Nanodisc-captured or detergent-solubilized receptors like the kappa opioid receptor (KOR) or GPCRs like the β2-adrenergic receptor (β2AR), the Cannabinoid receptor CB2, and the Adenosine A2A receptor have each been shown to lose binding activity within minutes at room temperature. The Gator Plus+ allows you to run these assays at scale at 10°C, which can improve their stability and assay lifetime.

Cooling to 10°C can also help to limit evaporation in long experiments, which normally causes instability by increasing reagent concentration. Below is an example: a 10 µg/ml biotinylated ProA was bound to SA XT biosensors in K buffer (which contains BSA) for 16 hours at 10°C, transitioning to new buffer wells of a 96-well plate every few hours with no significant deviation due to BSA aggregation over time. Meanwhile at 25°C, an upwards drift was observed after ~8 hours (not shown). Why? At 25°C, more than 50% had evaporated at the end of the 16-hour experiment, doubling the BSA concentration in the well, which could explain this drift in the control trace. However, at 10°C, there was no measurable loss of volume and minimal upwards baseline drift.

2. Resolving Fast-On/Fast-Off Interactions

Many biological interactions have evolved to be weak, with fast on- and fast off-rates. In a previous blog post, we talked about lowering assay temperature to below 25°C to better assess Fcγ receptor kinetics. Using this method, we can better capture the steep slopes at the beginning of association and dissociation. This is also particularly helpful for small molecules, peptides, and other micromolar-binders.

This works because molecular diffusion and reaction rates follow Arrhenius kinetics – lowering the temperature slows both association and dissociation in a predictable way. When testing Fcγ receptor affinity, for instance, the Fc domain of each candidate molecule is tested against the same receptor. In these experiments, the objective is to observe whether an alteration to the Fc has taken place as a result of engineering the antibody, so as long as all candidates and references are assayed under the same assay conditions (i.e. at 20°C), any variation in K_D as a result of running at different temperatures can be safely ignored.

3. Characterizing interaction thermodynamics and binding mechanisms

Surface-based biosensor techniques like SPR have been used for thermodynamic analysis of antibody-antigen interactions for several decades ^{[1, 2]}, though the approach is less-commonly applied to BLI. However, using the wider temperature range available to the Gator Plus+ (from 10ºC to 40ºC instead of 25ºC to 40ºC), the distinction between enthalpy-driven and entropy-driven interactions becomes far easier to observe. After measuring kinetics and affinity at multiple temperatures, you can apply the Van ‘t Hoff equation to extract the enthalpic (ΔH) and entropic (ΔS) contributions to binding. This is done by plotting 1000/T (the inverse of absolute temperature in Kelvin) on the X axis, and the natural logarithm of the equilibrium dissociation constant, ln(K_D), on the Y axis. The slope of the resulting line is proportional to enthalpy and the Y-intercept to entropy, as described by the Van ‘t Hoff equation:

Here’s a real-world example: Anti-HER2 (Trastuzumab) binding to HER2. We assessed the interaction of this binding pair across a range of temperatures from 10-40°C as shown in the sensorgrams below

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These results align well with established literature^[3]. To use these in-house results as an example of thermodynamics calculations possible with a BLI instrument, we can plot the K_D values in Van’t Hoff format to obtain the graph below

As seen on the Van ‘t Hoff plot, the interaction has a negative slope, meaning it is exothermic, releasing heat upon binding, which is why affinity decreases as temperature increases. After curve fitting, we can calculate that the enthalpic contribution is ΔH = −34.0 kJ/mol (multiply that slope number by R) and the entropic contribution is ΔS = +47.0 J/mol·K.

Does this make sense? Using the Gibbs free energy equation at 25°C, we can work back to see if our calculations from the fitting align with the results we obtained on the BLI instrument:

The experimental value of 3.74 nM at 25°C aligns well with the result from the Van‘t Hoff plot of 3.84 nM.

The trend line was fit to data from 15°C and above, as the 10°C point deviates noticeably from the linear relationship. That deviation can be mechanistically informative. Using the approach of Guardiola et al, we can use the Eyring equation to identify the individual activation enthalpies and entropies for the association (k_on) and dissociation (k_off) steps separately^[4]. When we plot ln(k_on/T) and ln(k_off/T) against 1000/T, the slope of each line gives the activation enthalpy ΔH‡ for that step (binding or unbinding), and the difference between them should equal the overall binding enthalpy.

Importantly, the 10°C data point deviates from the linear Eyring relationship in k_off but not in k_on, meaning that at this low temperature, the complex dissociates far more slowly than expected but association is unaffected. Arrhenius analysis of the same data yields equivalent activation enthalpies with fewer theoretical assumptions^[1], and both analyses lead to the same conclusion about the anomalous behavior at 10°C.

These findings are explained by SPR results^[5] that suggest antibody binding proceeds in two sequential steps: an initial diffusion-limited encounter, followed by conformational rearrangement into the final stable complex. SPR and BLI detect both steps together as a single observed binding event, but the two steps have very different temperature sensitivities. In this case, at 10°C, the fully docked complex is disproportionately stabilized, which is a signature of a non-zero heat capacity change (ΔCp) upon binding, consistent with burial of hydrophobic surface area at the antibody-antigen interface. Between 10°C and 15°C, this additional stabilization diminishes and the interaction follows the expected linear temperature dependence.

Not all antibodies follow this trend. Whether affinity rises or falls with temperature depends on whether binding is driven primarily by enthalpy or entropy, which in turn reflects the structural character of the antibody–antigen interface. Unlike Anti-HER2, whose affinity decreases with temperature, some antibodies have been shown to bind more tightly at febrile temperatures, suggesting entropy as the dominant driving force^[6].

These considerations become especially important in the design of multispecific antibodies. When a single antibody must sequentially bind two different targets, the thermodynamic cost of the first binding event constrains what is available for the second. Specifically, if binding the first target induces large conformational changes, the antibody may have reduced thermodynamic capacity to reorganize the rest of the structure to engage the second target. Temperature-dependent kinetic analysis of each binding arm can flag these incompatibilities and can guide the selection of binding arms whose thermodynamic profiles are mutually compatible.

The Gator Plus+: 10°C to 40°C, Peltier-controlled

The Gator Plus+ is the first BLI instrument to offer controlled cooling down to 10°C, with precise temperature regulation across the full 10-40°C range. This capability is built into the standard 8-channel 96- or 384-well plate compatible instrument.

Key features for low-temperature experiments include:

• Precise Peltier-based temperature control with consistent baseline stability at all temperatures
• Full kinetics and affinity measurement at any set temperature
• Access to Gator Bio’s high-performance biosensor tips – regenerate your HIS XT or Strep-Tactin biosensor and re-use them for each temperature band

Getting Started

Low-temperature BLI is a versatile tool, but like any technique, it benefits from thoughtful experimental design.

• Buffer osmolality and viscosity change with temperature. Running matched buffer blanks at each experimental temperature is essential – you can’t use a 10°C blank for reference subtraction for your 40°C sample. Similarly, you can’t transition between plates that are at different temperatures and expect a consistent baseline.
• If doing multiple temperature points, start with low temperature. Not only does this ensure the best sample stability and performance, but the plate heats faster than it cools because heat rises.
• Allow time for biosensor hydration and plate equilibration. At 10°C, a typical equilibration time of 10 minutes is sufficient to bring the sample plate to the target temperature before data collection begins.
• Pay attention to strong deviations in K_D – the results of the Anti-HER2/HER2 interaction above show that changing the temperature by a few degrees shouldn’t dramatically affect a typical antibody-antigen interaction K_D. However, if temperature changes the kinetics dramatically, the change could be caused by stability (i.e. aggregation of the analyte at high temperatures or increased NSB at low temperatures), not a decrease in affinity.

Our applications team has run these assays across GPCR targets, Fc receptor panels, and antibody bispecifics. If you have a target that has been difficult at room temperature, reach out to arrange a discussion.

The Gator Plus+ is available now. Contact us at gatorbio.com/quote to learn more.