Technical Support Corner
Tips and Tricks for Kinetic Assay Optimization

Pui Seto and Yixin Lin, Technical Support Team

At Pall ForteBio Technical Support, we often get questions about various aspects of running kinetic assays. As with any assay, optimization in kinetic analysis is extremely important. Taking the time for proper assay development ensures that resulting kinetic data doesn't end up fraught with artifacts and that binding and affinity constants are meaningful. We've listed some suggestions here that we commonly provide customers for optimizing kinetic assays on the Octet and BLItz systems.

  1. Choose the appropriate biosensor format based on the Biosensor Selection Guide, reagent availability, and prior knowledge of the interaction.
  2. Choose the appropriate assay buffer based upon reagent availability, stability and prior knowledge of the molecules being studied. For example, for large biomolecule and peptide kinetics, use a buffer that has some carrier protein and detergent to maintain reagent stability, prevent loss of protein to tube or well surfaces, and minimize nonspecific binding (NSB). For small molecule kinetics you should avoid using carrier protein in the assay buffer.
  3. To reduce non-specific binding (NSB), add carrier protein, detergent, and/or salt as needed. Addition of 1% BSA and up to 0.05% Tween 20 to PBS buffer (or other buffer system) can be very effective at reducing NSB. Use this buffer for all steps, including hydration of biosensors. This same buffer should also be used for dilution of ligand and analyte. Please consult Pall ForteBio Application Note 14, Biomolecular Binding Kinetics Assays on the Octet Platform, for additional suggestions on NSB reduction.
  4. When possible, analyte concentrations should bracket the expected KD value, ranging from 10X above to 10X below the KD with 2-fold dilutions in between. If the KD value is not known, perform a scouting experiment with a few wide-ranging analyte concentrations to estimate the KD. Always perform global fitting when calculating KD values.
Octet

BLItz

  1. Avoid using bivalent or multivalent molecules (including antibodies) as analyte in solution since avidity or bridging effects can influence the dissociation rate constant and make the interaction appear tighter than it really is. Multivalent molecules should be immobilized on the biosensor whenever possible for accurate affinity constants. If bivalent or multivalent partners must be used as analyte, reduce the loading density of the immobilized ligand as much as possible (while still maintaining adequate association signal) in order to minimize avidity effects.
  2. Using 100–300 nM ligand for loading to achieve 0.5–1 nm response is a good starting point for most large molecule kinetics experiments. However, for small molecule kinetic experiments, load the biosensors to saturation with target protein, achieving at least an 8 nm response if possible.
  3. A general guideline for assay step times:
    • Initial baseline — 60 seconds
    • Ligand (target) loading — 300 seconds
    • Baseline — 120s
    • Association — 180 to 300 seconds
    • Dissociation — 600 to 900 seconds.
    These suggestions are only a starting point — each step time should be optimized as needed.
  4. A loading negative control biosensor (without any ligand loaded, dipped into analyte directly) and a reference sample (loaded with ligand, then dipped into buffer instead of analyte) should be included early in assay development. The loading negative control will reveal any non-specific binding. The reference sample should be used for baseline drift subtraction. A reference sample should be included in every kinetic assay.
  5. Whenever possible, immobilize the smaller binding partner on the biosensor surface and dip into the larger molecule in solution for the association step to maximize binding signal.

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You can reach us at Pall ForteBio Technical Support: call 1.888.628.3875 or e-mail inquiries to
fortebio_support@pall.com.