ProteOn XPR36, User Manual

Page: 147 / 288

Analyzing Data
129
In the latter instance, the two analytes compete and the higher
affinity analyte replaces the data with the lower affinity
• Heterogeneous ligand — interaction where one analyte binds two
different ligand species. When ligands are bound to the surface of
the chip, it is possible that they may be bound in different
orientations, resulting in multiple ligands. Additionally, the use of
polyclonal antibodies or the study of post-translational
modifications could give rise to heterogeneous ligands. In this
case, because each ligand-receptor complex should have separate
affinities, the resultant sensorgram reflects the sum of two
independent reactions with two sets of rate constants
• Two states — interaction where the conformation changes upon
ligand-analyte binding. The two states model accounts for a
complex that changes shape after interacting with the analyte.
There are three states for the ligand and analyte to take in this
model: 1) ligand and analyte free in solution, 2) ligand-analyte
complex, and 3) conformational change of the ligand-analyte
complex. It is equal for the ligand-analyte complex to form the
conformational change or to return to free molecules in solution.
However, in order to release the ligand, the complex must change
back to the ligand-analyte complex
• Langmuir with drift — langmuir model with an extra parameter for
fitting data with linear baseline drift. When the ligand is not
covalently bound to the surface of the chip, the interaction may
encounter drift. This is common in His-tag experiments using NTA
or with unpurified monoclonal antibodies using a capture antibody.
In these cases there is a capturing agent covalently bound to the
surface of the chip and a ligand (like His-tagged protein or
monoclonal antibody) bound to the capture molecule. The analyte
is flowed in solution across the surface of the chip. As the analyte is
flowed across the surface and subsequently washed for the
dissociation phase, the ligand may leach from the surface of the
chip at the same time. Note that the Langmuir with drift model
calculates only a linear drift that is constant with time. Therefore, it
can be applied to a very slow leaching of the ligand from the
surface