DRX
switchBUILD Software
There are two basic approaches to detect a conformational change:
- The first approach is to include a sizing measurement into a kinetic. This way, the association and dissociation kinetics of the test compound to the ligand molecule will be recorded and at equilibrium (in between association and dissociation) a sizing or stopped flow experiment will be performed. This approach is particularly suitable, if you are also interested in the kinetic rate constants and the affinity constant of the interaction between test compound and ligand.
- The second approach utilizes the sizing tool for His6-tagged proteins in switchBUILD. In this case, the His6-tagged ligand molecule is pre-incubated with the compounds to be tested, immobilized on the switchSENSE® chip and a sizing experiment is performed. Especially when testing very weakly binding compounds (e.g. fragments), this strategy is advisable as very long pre-incubation times can be chosen.
The switchBUILD kinetic tool helps to easily set up an optimized taskflow to analyze the interaction of interest and automatically suggests concentration ranges and measurement times based on the estimated affinity of the interaction partners.
When no affinity constants are known, an educated guess of the affinity range helps to set up your assay. This can be done, for instance by choosing an interaction with a comparable molecule class (e.g. antibody) from the provided drop-down list. Based on this estimate, an affinity scouting can be carried out. To do so, choose a high dilution factor (at least 3x) and at least three concentration steps to cover a possibly wide concentration range. This way, it is usually possible to roughly determine the affinity range which can be narrowed down in further experiments.
For kinetic experiments, choosing the appropriate flow rate can be a crucial factor for determining the correct affinity. This can possibly lead to measurement artifacts that are caused by insufficient flow rates. During the association phase, a too slow flow rate can be the cause for mass transport limitation effects. On the other hand, during the dissociation phase an inadequate flow rate can result in re-binding effects. Both these effects should be avoided to obtain high quality kinetic data. Thus, the general rule is to work at a comparably high flow rates (e.g. > 100 µl/min) whenever the experimental circumstances allow. However, there are certain limitations to this, as sample and buffer amounts are often of limited availability.