In this study, we aim to purify flap endonuclease 1 or FEN1, a crucial DNA replication protein using affinity chromatography. This will allow us to investigate FEN1's function in DNA replication and repair in a controlled in vitro environment, advancing our understanding of its functions and biological roles. Various types of chromatography are used to purify DNA binding proteins, the most common being affinity chromatography.
Within affinity chromatography, immobilized metal affinity chromatography is one of the strongest tools to purify His-tagged proteins since the interaction between the His-tagged and the metal ion, usually nickel or cobalt is very strong. Two crucial factors for successful protein purification process are protein yield and protein purity. Depending on the protein's intended use, either yield or purity might be preferred over the other.
Our findings show that selecting the appropriate resin for the purification of His-tagged proteins might yield results desirable for the proteins downstream applications. To begin, fit the columns to a clamp stand as straight as possible. Plug the bottom of each column with a stopper.
Add one to two milliliters of starting buffer to the bottom of each column to prevent air bubbles during column packing. Then open the top of the columns and pour in 10 milliliters of each slurry continuously against a metal spatula or glass rod held at the rim of the column to prevent air bubbles. Remove the stopper from the column and connect the column's bottom to one end of the tubing.
Attach the other end of the tubing to the UV port of the FPLC system. Verify that the bed support housing is pulled into the flow adapter body. And slide the flow adapter body onto the top of the column with the cam latch in position 1, lower the flow adapter.
Then switch the cam latch to position 2. Connect the tubing to the system and start the buffer flow for one to two minutes to eliminate air from the tubing and prevent air bubbles during packing. Afterward, switch the latch back to position 1, lower the flow adapter smoothly into the buffer, avoiding air bubbles, and switch the latch back to position 2.
After switching the latch from position 2 to 3, tighten both the cam base and the locking ring. Then start the buffer flow at 10 milliliters per minute for three minutes or until the resin is well-packed. After packing the FPLC column, connect the tubing from the top of the sample loop to Loop E on the FPLC system.
Attach the tubing from the bottom of the sample loop to Waste 2 on the FPLC system. Then insert one end of the tubing into the column port of the FPLC system and place the other end in the waste bottle. Insert the tubing from Pump A into filtered double distilled water.
Next, open the software. Click on the System Control tab. Double click on the Pump Settings and select System Pump Inject Loop.
Click on the Flow Rate Settings and set the flow rate to 10 milliliters per minute. Enter 0%in the percent B box. Then start the flow to flush the sample loop with water to push the sliding seal downwards.
Once the sliding seal reaches the bottom of the sample loop, stop the flow. Then switch Pump A from double distilled water to the starting buffer. Connect the tubing from the top of the sample loop to Waste 2 and the tubing from the bottom to Loop E.Leave the tubing from the column port in the waste and start the flow again at 10 milliliters per minute.
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 5 CV.For Illusion, 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 eluded protein.
