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Binding Assays
Commonly asked questions of foundry
Why don't I always get KD values for my proteins?
Why don't I always get KD values for my proteins?
Our assay works by loading your proteins, referred to as ‘constructs,’ onto BLI sensors, where their interaction with a specific target protein, called the ‘analyte,’ is measured over time. The association curve (binding response when the sensor is exposed to a solution with a known analyte concentration) and the dissociation curve (response when the sensor is moved to a solution without analyte) are used by our software to calculate the equilibrium constant (KD) and the kinetic constants (ka - association rate, kd - dissociation rate).For reliable binding data, the protein must express as a soluble protein.We categorize expression levels as None, Low, or Normal.
- None: No binding test can be performed, and the binding strength will be marked as Unknown.
- Low: Binding events may be detected, but with a low signal-to-noise ratio, making it difficult to accurately estimate the KD. In these cases, the binding strength is usually labeled as Weak, without a precise KD value. In most cases, binding is quantifiable, and a KD will be displayed.
What is the normal range of KDs you can quantify? Can you go beyond that range? Are there cases where not the full range is accessible?
What is the normal range of KDs you can quantify? Can you go beyond that range? Are there cases where not the full range is accessible?
Under normal conditions, our system can quantify KD values in the range of 0.1 nM to 10 μM. Toward the extremes of this range (0.1–1 nM and 1–10 μM), the accuracy may decrease because there is less data to fit when dissociation is either extremely fast or extremely slow.We offer flexibility in protocol parameters (e.g., interaction times and analyte concentrations) to tailor experiments for specific needs. For example, we can focus on tight binders (KD of 10 nM or better) but may sacrifice the ability to observe weak interactions, or we can focus on weak interactions (1–50 μM), but this may limit the ability to precisely calculate KDs for stronger binders.In cases where an analyte shows high non-specific binding, we must reduce the analyte concentration, limiting our ability to detect very weak interactions.
What do the binding labels mean?
What do the binding labels mean?
What do the binding labels mean?
- True: A clear binder, showing binding curves significantly above our negative controls.
- False: A non-binder, where the construct expresses, but the binding curves are at or below the expected signals for non-binders (based on negative control curves). The threshold between true and false binders can depend on analyte size (smaller analytes may lower detection limits) and non-specific binding (higher non-specific binding may raise detection limits).
- Unknown: Used when there is no or low expression, or when binding curves are slightly above negative controls but not significantly so.
Binding Strength:
- Strong: KD below 50 nM.
- Medium: KD between 50 nM and 1 μM.
- Weak: KD above 1 μM.
- Unknown: Typically used when there is no expression or when low expression prevents accurate KD measurement.
How is experimental data processed and how are curves fitted?
How is experimental data processed and how are curves fitted?
For each BLI probe, the instrument records time and wavelength shift data at 5 Hz (one data point every 0.2 seconds). Our software uses this data, along with the instrument protocol file (which details the steps and durations of each stage) and the mapping file (which identifies the contents of each well) to associate the customer’s protein sequences with specific run results.In a typical run, a sequence is tested against one or more analyte concentrations. A 1:1 binding model is applied to the data, and if multiple concentrations are used, global fitting is employed.
How can I interpret KD values in binding screening experiments?
How can I interpret KD values in binding screening experiments?
In protein-affinity characterization, constructs are tested against multiple analyte concentrations to ensure that at least two concentrations are close to the KD, where the signal-to-noise ratio is optimal. In contrast, binding screening experiments focus on distinguishing binders from non-binders and providing a rough estimate of binding strength (strong, medium, weak). This is done using only one or two analyte concentrations, so KD estimates in screening experiments carry higher uncertainty than in full affinity characterizations.
Why do some proteins express poorly or not at all?
Why do some proteins express poorly or not at all?
- Solubility issues: Some protein sequences tend to self-aggregate and precipitate out of solution, preventing them from being loaded onto sensors. This is largely a property of the protein sequence itself, for example when the protein has hydrophobic patches on the outside that cause it to stick to other proteins in order to not be exposed to the surrounding solution (the expression buffer).
- mRNA structure and interference: In any system that uses natural cellular machinery (e.g., ribosomes), mRNA secondary structures can prevent proper translation. Additionally, some proteins may interact with the machinery, causing premature translation termination. By default, our codon optimization software aims to minimize the stability of the mRNA secondary structure for all of your proteins but this might not be possible in all cases, depending on the amino acid sequence of your proteins.
What are your main binding assay services, and how do they work?
What are your main binding assay services, and how do they work?
Our primary binding assays are run using Biolayer Interferometry (BLI) and Surface Plasmon Resonance (SPR). We offer two main tiers of service:
- Protein Binding Screening: This is a rapid, cost-effective assay ideal for initial screening of new designs. It uses two different analyte concentrations to quickly categorize your variants into binding strength buckets (e.g., Strong: KD <50 nM; Medium: KD 50 nM – 1 µM; Weak: KD > 1 µM).
- Protein Affinity Characterization: This is a more in-depth assay designed for optimizing known binders. It uses a full concentration series (typically five or more concentrations) to determine a precise KD value, along with kon and koff rates.
Can you test for binding specificity, cross-reactivity, or off-target binding?
Can you test for binding specificity, cross-reactivity, or off-target binding?
Yes, we offer several assays to assess binding specificity. We can perform simple cross-reactivity tests to confirm that your binder interacts with its intended target but not with a closely related ortholog or other specified protein.For more general specificity profiling, we offer polyspecificity assays that measure non-specific binding against complex mixtures, such as baculovirus particles (BVP), or abundant serum proteins like bovine serum albumin (BSA) or human serum albumin (HSA). More complex assays using full serum are currently in development.
How do you handle high non-specific binding (NSB)?
How do you handle high non-specific binding (NSB)?
We proactively screen every new target protein for non-specific binding (NSB) to our sensor surfaces before starting an experiment. If high NSB is detected, we will troubleshoot by trying different buffer conditions (e.g., adding more blocking agents) or by sourcing the target from an alternative supplier, as production methods can influence a protein’s behavior.High NSB can sometimes cause binding curves to show a negative shift. This happens when the reference sensor (without binder) binds more non-specifically to the target than the test sensor (with binder), causing the reference-subtracted signal to drop below zero.
Why do my binding curves sometimes look unusual or bi-phasic?
Why do my binding curves sometimes look unusual or bi-phasic?
It is not uncommon to observe complex or “non-standard” binding kinetics. Bi-phasic curves, often characterized by an initial fast binding phase followed by a slower one, can be indicative of a multi-step binding mechanism, such as a conformational change in the target or binder upon interaction. This has been observed even with monomeric targets.While our fitting software provides a good approximation in these cases, and the relative affinity ranking across your variants should remain reliable, a full kinetic series with a wider range of concentrations may be needed to accurately model the complex mechanism and determine a precise KD.
Do you offer competitive binding or inhibition assays?
Do you offer competitive binding or inhibition assays?
Yes, we can perform several types of competition assays. We can run neutralization or inhibition assays (e.g., for IC50 determination) where your designed protein competes against a natural ligand for binding to the target.We can also configure experiments to determine if a binder’s access to its binding site is blocked by other molecules, which is useful for epitope binning or mechanism-of-action studies.