Enzyme Kinetic Assay - worked example

Plan the design

This example focuses on creating a platemap that varies an enzyme against a substrate for 2
different buffers on a 384-well plate.

  • Buffer A and Buffer B are kept constant at 5mM
  • Substrate is an n-fold gradient going from 0.3nM to 300nM for each buffer, with a control on
  • Enzyme has 4 different concentrations: 10nM, 20nM, 50nM and 100nM
  • Use a 384 well plate with 4 replicates of each condition

Create a new file

Create a new file either when you start the software or by clicking on the ‘new design’ button:

Select the correct plate dimensions and final assay volume

Add components

Add all 4 components, type in the names and their stock concentrations and select the required
units from the drop-down menu.

Create the first region

For the first region, only half of the plate is needed. First, click on the resizing icon.

Then, change the region size by dragging the right-hand side edge of the region to the middle:

Click into the region to change the name of the region.

Add components to the region

To add the components to the region, click into the region and press ‘+ Add Component’.

Then (1) select the component from the drop-down menu, (2) choose the direction of the gradient which is in this case ‘horizontal’ and (3) select which gradient type, here ‘n-fold’.

To further define the gradient, either open the gradient editor dialog by clicking on ‘Edit…’ or
change the required parameters in the editing area above the grid:

Change the concentration start to 0.3 and the end to 300 and make sure a zero is added by ticking the tickbox.

Then add the next component to the region:

For the substrate, change the direction to ‘vertical’ and select ‘ad-hoc’ to type in the required
concentrations. To do this, open the Gradient Editor by clicking on ‘Edit…’

In the gradient editor window, insert the concentrations. The grid UI in the background will give
direct visual feedback.

Add point copies by increasing the ‘Point Copies’ to 4 and click OK.

Finally, add the buffer as a constant and change the concentration if necessary in the control
above the platemap grid.

Create the second region based on the first

Because the second region is identical apart from the Buffer component, copy and paste the
region either by right-clicking the mouse or using Ctrl+C and Ctrl+V to paste the copy in the
regions area.

Once the region is copied, change the region name and change Buffer A to Buffer B.

Then, go back into the region editing mode

And shift the entire region to the right by dragging it with the mouse.

Export the design

Click on the Export tab to get an overview of the finished design and calculated volumes.

To check concentrations, select ‘Concentrations’ from the drop-down menu above the component

On the bottom left of the export tab, the reagents list shows how much is needed of each stock.
Note that in this example the Substrate stock of 600nM is too high to dispense the lower
concentrations. The software calculates the required intermediate stock at 9.7nM. By changing the final assay volume, the intermediate stock will change as well.

Next article - Defining regions