I. Characteristics of the Pull-down Assay

    The pull-down assay is an in vitro method widely used to detect or confirm interactions between various proteins. This method is similar to immunoprecipitation, in that it uses an affinity ligand to capture interacting proteins. The difference between the two methods is that immunoprecipitation uses immobilized antibodies to capture protein complexes, whereas the pull-down method uses purified and labeled proteins as “baits” to bind any interacting proteins.

II. Principles of the Pull-Down Assay

    This method involves first immobilizing a labeled protein (bait) onto a tag-specific affinity ligand to create an affinity support for capturing and purifying other proteins (prey) that interact with the bait. Bait and prey proteins can be obtained from various sources, such as cell lysates, purified proteins, expression systems, and in vitro transcription/translation systems. Once the prey proteins have been incubated with the immobilized bait protein, the interacting complexes can be eluted using an elution buffer appropriate for the specific affinity ligand. Each experiment requires appropriate controls to demonstrate that the observed interactions are not artifactual. After the pull-down experiment, the protein components are resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then visualized by gel staining or Western blot analysis.

III. Reference Experimental Procedures

3.1 Preparation of Cell Lysates

1. Place eukaryotic cells in a cell culture dish and incubate overnight in a 37°C CO₂ incubator.

2. Transfect the cells with a plasmid containing the target gene and an appropriate transfection reagent, allowing sufficient time for optimal protein expression (16–24 hours is typically a good range).

3. Place the culture dish on ice to cool the cells, then wash the cells with 1× PBS. Add 2 mL of pre-chilled PBS and collect the cells using a cell scraper.

4. Centrifuge at 4°C and 80 × g for 5 minutes.

5. Resuspend the cells in 200 μL of RIPA buffer containing an inhibitor.

6. Incubate on ice for 20 minutes, gently mixing every 5 minutes.

7. Store the prepared cell extract at –80°C.

8. Thaw the prepared cell extract prior to the pull-down experiment. Centrifuge at 4°C, 17,000 × g for 20 minutes. Use the supernatant as the bait.

3.2 Pull-down Assay Using Cell Lysate as Bait.

1. Transfer 120 μL of Ni-NTA agarose beads to a gravity flow column.

2. Centrifuge at 4°C, 1000 × g for 1 min.

3. Add 400 μL of distilled water to the column.

4. Centrifuge at 4°C, 1000 × g for 1 min.

5. Mix 50 μg of His-tagged protein (bait) with 400 μL of equilibration buffer, and load onto the column.

6. Incubate for 1 hour with stirring at 4°C, then let stand on ice for 10 minutes.

7. Centrifuge at 4°C, 1000 × g for 1 minute.

8. Mix 200 μL of cell extract with 200 μL of equilibration buffer and load onto the column.

9. Incubate for 1 hour at 4°C with stirring, then let stand on ice for 10 minutes.

10. Centrifuge at 4°C, 1000 × g for 1 minute. Maintain the flow for analysis.

11. Add 400 μL of equilibration buffer to the column to wash it.

12. Centrifuge at 4°C, 1000 × g for 1 minute.

13. Add 400 μL of equilibration buffer containing 50 mM imidazole to the column to wash it. Reserve the first wash for analysis.

14. Centrifuge at 4°C, 1000 × g for 1 minute.

15. Repeat steps 14 and 15 three times, then proceed to step 16. Store the final wash solution at 4°C for analysis.

16. Load 80 μL of elution buffer onto the column and incubate at 4°C for 10 minutes.

17. Centrifuge at 4°C, 1000 × g for 1 minute, and retain the eluted fraction.

18. Repeat steps 16 and 17 with the eluted fraction. Keep the eluted fraction at 4°C for analysis.

3.3 Pull-down Assay Using Purified Protein as the Prey

1. Incubate 50 μg of His-tagged bait protein with 50 μg of purified prey protein in a total volume of 400 μL of equilibration buffer for 2.5 hours, with stirring at 4°C.

2. Add 80 μL of Ni-NTA agarose beads to a gravity-flow column.

3. Equilibrate the column with 400 μL of equilibration buffer containing 20 mM imidazole.

4. Centrifuge at 4°C, 1000 × g for 1 min.

5. Load 400 μL of the incubated bait and prey proteins onto the column. Incubate on ice for 10 minutes.

6. Centrifuge at 4°C, 1000 × g for 1 min. Keep the eluate at 4°C for analysis.

7. Add 400 μL of equilibration buffer supplemented with 20 mM imidazole to the column to wash it.

8. Centrifuge at 4°C, 1000 × g for 1 min. Store the first wash fraction at 4°C for analysis.

9. Repeat washing steps 7 and 8 four times, and store the final wash supernatant at 4°C for analysis.

10. Add 200 μL of elution buffer to the column and incubate on ice for 10 minutes.

11. Centrifuge at 4°C, 1000 × g for 1 min. Store the eluted fractions at 4°C for analysis.

3.4 SDS-PAGE and Protein Composition Analysis

1. Add 5 μL of lysis buffer to 15 μL of the protein fraction and concentrate fourfold. Heat at 100°C for 3 min, centrifuge in a microcentrifuge for 30 s, and collect the supernatant.

2. Load 10 μL of the eluted protein and 5 μL of protein marker onto an SDS-polyacrylamide gel.

3. Run the gel at 100 V for 15 minutes, then increase the voltage to 180 V until the leading edge of the dye reaches the bottom of the gel.

4. Identify interacting proteins by immunoblotting or blue Coomassie staining.

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