Kinomics MSK ISA Application
(ISA) utilizing Multi-Step Kinetics (MSK) on Biacore
- This true initial base
rate is directly proportional to the analyte
(enlarge, 450 kB, pdf )
Determination of the inhibition
constant KI (in solution)
between analyte A and inhibitor I that are
mixed prior to the MSK study on a
Biacore system where the equilibrated solutions
are checked for the free analyte A against
ligand L (immobilized on the sensor) with
regard to non-inhibited analyte references
in order to identify inhibitors of interest.
A data simulation.
· Mix analyte:inhibitor (A:I
= 1:1 reaction type) at different (≥5)
concentrations; especially in biosensor
capture assays, prepare non-inhibited analyte
· Run MSK (w/o intermediate
regenerations) with L immobilized (or captured):
- Vary contact times (allow low analyte
concentrations to bind longer; see 1).
- Inject the ≥5 pre-mixed inhibited
samples, each followed by a dissociation.
- In the same way, inject non-inhibited
reference samples (right in 1); the # of
these may be less or, in case the assay
is highly reproducible, skipped at all.
· Non-linear fitting and evaluation
of data with MSK software incl cross-checks:
- Import affinogram (black curves in 1)
and fit by a single click within 2 s (red).
- Exclude inhibited samples from evaluation
(even so, they are kept calculated;
shown yellow in 2) and, if indicated, omit
outliers of non-inhibited references.
- Evaluated references yield KD
of A:L (here 6 · 10-8
mol L-1) incl kinetics; the green
traffic lights (3) indicate an excellently
cross-checked reference result.
from initial association rates (non-linearly
extrapolated back to baseline):
In MSK from Kinomics, this rate is readily
available for each single step!
- The fit of the non-inhibited samples' rate yields
the reference slope (red in 4).
- From the suppressed rate of the inhibited pre-mixes
(yellow dots in 4), their actual free A concentration
is calculated with regard to the reference slope.
- Thus, the equilibrium concentration (ceq)
of relevant compounds (A, I, AI) in the pre-mixes
are known: KI is calculated according
to the mass action law (5).
· Intuitive: The 50 % inhibition
position of A (in 4) indicates the KI
at a glance or, in other words, strong inhibitors
suppress the rates more strongly.
· In comparison with others, identify
the inhibitor(s) of your interest.
Done. Fast, easy, and intuitive. It's unique
it's from Kinomics.
Published online 11/25/2009 on www.kinomics.com
© Kinomics GmbH 2009
Kinomics MSK ISA Application Note