To begin, pack the FPLC column and prepare the sample loop for sample loading. Now go to the software. Double-click on the pump settings and select Manual Load Loop.
Now connect the tubing from the column port to the tubing on top of the flow adapter. Unscrew the tubing from loop E and place it into the waste container. Then using a 30-milliliter syringe, draw up the sample and attach the syringe to the injection port adapter.
Screw the syringe into the injection port and inject the sample, ensuring it is visibly injected into the sample loop. Now plug the injection port with the column cap. Connect the tubing from the bottom of the sample loop to the loop E port.
Then place the tubing from pump B into the elution buffer. Navigate to the Home tab and select New Method. After that, select the Method Settings tab on the left column.
Go to the Column Type dropdown menu and select Custom. Enter the column volume as five milliliters and the required flow rate for the run as one milliliter per minute. Navigate to unit selection and select CV, column volumes, as the method base unit.
Verify that inlet A is set to buffer A1 and inlet B to buffer B1.Then select the Method Outline tab on the left-side menu. Under Equilibration, set the equilibration volume to 10 CV.Under the Sample Application tab, set the sample volume by observing the sample loop. Navigate to Column Wash and set the wash volume to five CV.For Elution, set the gradient volume to 14 CV, starting from 0%buffer B to 100%buffer B.Save the method and start the run.
Ensure that the fraction collector starts at the position of the first fraction. Enter the run name and click Start. During sample application, switch the tubes currently going into the waste container to a clean beaker.
Collect the unbound proteins flowing out of these tubes as the flow through and label it as FT.After the run is complete, navigate to the Home tab and select Open Run. Open the run to view the chromatogram. Select Peak Integration on the top menu to mark the obtained peaks.
Select the Fractions tab to visualize the fractions containing eluted protein.
