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Make sure your sample works
When it comes to measuring binding affinity, the quality of your results depends on the quality of the source
material. The molecules you're investigating need to be in good shape — literally — in order to perform the
interaction correctly. For example, a protein's binding capabilities depend on its 3D structure. Things like
temperature, pH, buffer substances, or storage conditions can cause the protein to unfold, rendering it unable
to interact. Consider confirming your sample's integrity before you run your binding affinity measurements.
What tools are available to measure binding affinity?
Scientists use many different tools to measure binding affinity, although
most of them fall into one of two categories: qualitative methods and
quantitative methods. Many qualitative methods such as ELISAs, pull-
down assays, and gel shift assays work by immobilizing one molecule to
a substrate and applying another molecule (typically containing a label or
reporter tag) to it. If the two molecules bind, the labeled or tagged molecule
is bound to the substrate via the immobilized molecule and can then be
detected on the substrate. These methods merely provide a yes/no answer as
to whether binding occurred and may therefore be suitable for labs that only
occasionally analyze interactions or are examining interactions at a very
superficial level.
In contrast, quantitative methods provide a scalar readout of binding
affinity, meaning that they reveal the strength of the interaction.
Some of these methods work by immobilizing a binding partner to
a surface and presenting the test partner to interact with it. Others
work in solution instead. The physical phenomena used as readouts
differ greatly between them, leading to quite different experimental
designs and instrumental setups.
In this section, we'll walk through the most common biophysical
technologies and their strengths and weaknesses in terms of
throughput, speed, sensitivity, and ease of use.
