PDA - Pull-down Assay

The Pull-down assay is an in vitro technique used to detect interactions between proteins using a bait protein. This assay is a form of affinity purification that is similar to immunoprecipitation and is useful for confirming and identifying previously unknown protein-protein interactions.

Bait Protein:

There are two ways of generating bait proteins for Pull-down assays, one being, linking an affinity tag to proteins that have been traditionally purified, second being expressing recombinant fusion-tagged proteins.

• Fusion Tags:

• Glutathione S-transferase (GST)

• Poly-histidine (polyHis or 6xHis)

• Biotin

• Affinity ligand:

• Glutathione

• Nickel or cobalt chelate complexes

• Streptavidin

This method relies on immobilising fusion proteins on a solid phase, known as beads. The bait protein is captured by ‘prey’ proteins and “pulled down” when the target-protein-binding beads are mixed and allowed to interact with the cell lysate.

Material Required:

• Wash buffer (50ml)

• Lysis buffer (10ml)

• Glutathione Sepharose Beads

• 2x HNG (100ml)

• PBS

Protocol for GST Pull-down Assay:

Immobilisation of Protein onto Glutathione Beads:

1. Mix 1ml of PBS, 200ug of protein and 40µl of resuspended glutathione beads.

2. Incubate for up to 1 hour at 4°C.

3. Centrifuge the beads for 30 seconds, aspirate supernatant and add 1ml PBS.

4. Wash step to be repeated 2x.

5. Resuspend the beads in 200µl final volume of buffer.

Preparation of Bacterial Cell Lysate:

1. Prepare lysis buffer with detergent.

2. Prepare and treat cells.

3. Wash cells 2x with ice-cold PBS (10ml for a 10cm dish).

4. Add 1ml lysis buffer and scrape the cells with a cell scraper, after transfer to a 1.5ml Eppendorf tube.

5. Incubate for 30 minutes at 4°C.

6. After, centrifuge at 4°C for 10 minutes.

7. Once complete, transfer all supernatant to a fresh tube.

Preparation of affinity beads:

1. First, calculate the volume of lysate to be used per pull-down (for example, for > 1ml of lysate and 3 pull-downs, you can use up to 333µl of lysate per pull-down.

2. Calculate the amount of buffer to resuspend the beads in (If you want to use 300µl of lysate you would resuspend the beads in 300µl of buffer).

GST Pull-down:

1. Take out a 50µl aliquot of each lysate sample and add 50µl of 2x sample buffer as a lysate control.

2. Add the appropriate amount of lysate to each set of beads.

3. Incubate tubes at 4°C for 30 minutes.

4. Add 50µl of glutathione Sepharose beads to each tube to increase the bed volume of beads and to limit accidental aspiration of beads.

5. Wash each tube with 1x HNG five times at 4°C.

6. Add 20 - 40µl of 2X SDS Loading Dye.

7. Boil samples for loading on a gel.

8. Calculate what 0.5% Lysate Volume is and load 0.5% lysate and the entire volume of the pull-down.

9. Stain the gel with either silver stain or Coomassie.

10. Excise the protein bands from the gel for further processing.