Binding Assays
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?
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?
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?
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?
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?
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.
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