Pull-down

The pull-down assay is an in vitro technique used to detect physical interactions between two or more proteins and an invaluable tool for confirming a predicted protein-protein interaction or identifying novel interacting partners. This method typically involves the use of affinity purification with various wash and elution steps.
Pull-down approach uses a purified and tagged protein as a“bait”to bind any interacting proteins. The method consists of first immobilizing the tagged protein (bait) on an affinity ligand specific to the tag, creating an affinity support to capture and purify other proteins (prey) that interact with the bait. The bait and prey proteins can be obtained from multiple sources, such as cell lysates, purified proteins, expression systems, and in vitro transcription/translation systems. Once the prey proteins have been incubated with an immobilized bait protein, interacting complexes are eluted using an eluting buffer depending on the affinity ligand. Following a pull-down experiment, protein fractions are resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then visualized by gel staining or western-blotting detection.

MCE has not independently verified the accuracy of these methods. They are for reference only.

1. Materials

1.1 Preparation of Cell Lysate

Prepare all solutions with distilled water at room temperature and keep them at the indicated temperatures.

Eukaryotic cells；

plasmid containing the gene of interest fused to a specific tag；

transfection reagent；

1×PBS；

RIPA buffer ；

antiprotease cocktail: mix 1% (v/v) of protease inhibitor cocktail (HY-K0010), phosphatase inhibitor cocktail 2 (HY-K0022), phosphatase inhibitor cocktail 3 (HY-K0023) and PMSF (HY-B0496)。

1.2 Pull-Down Assays

1.2.1. 1 M Tris-HCl, pH 7.5 stock solution. Weigh 121.1 g Tris base and transfer to a 1 L graduated cylinder. Add water to 800 mL, mix, adjust pH with HCl, and make up to 1 L with water. Store at room temperature.

1.2.2. 5 M NaCl stock solution. Weigh 292.2 g NaCl and transfer to a 1 L graduated cylinder. Add water to 800 mL, stir, and adjust volume to 1 L with water.

1.2.3. Equilibrium buffer: 20 mM Tris-HCl, pH 7.5, 250 mM NaCl. Mix 1 mL 1 M Tris-HCl, pH 7.5 stock solution with 2.5 mL 5 M NaCl stock solution in a 50 mL centrifuge tube, and add water to a volume of 50 mL. Keep at 4℃.

1.2.4. Elution buffer: 20 mM Tris-HCl, pH 7.5, 250 mM NaCl, 500 mM Imidazole. Weigh 1.7 g Imidazole in 50 mL solution of equilibrium buffer. Keep at 4℃.

1.2.5. Purified His-tagged protein (bait).

1.2.6. Ni-NTA agarose beads: 6% beaded agarose (cross-linked), precharged with Ni²⁺ (Protino^® Ni-NTA Agarose). Store at 4℃.

1.2.7. 0.8 mL empty columns for gravity flow (Pierce^™ Centrifuge Columns).

1.2.8. Refrigerated microcentrifuge.

2. Methods

2.1 Preparation of Cell Lysate

2.1.1. Seed eukaryotic cells at 5x10⁵ in 10 cm cell culture dish and incubate overnight at 37℃ in CO₂.

2.1.2. Transfect cells with plasmid containing gene of interest fused to a specific tag with appropriate transfection reagent for time necessary for optimal expression of protein (16-24 h is usually a good range).

2.1.3. Cool cells by placing plates on ice, wash cells with 1×PBS. Add 2 mL cold PBS and harvest cells using cell scraper.

2.1.4. Centrifuge 5 min at 80×g at 4℃.

2.1.5. Resuspend cells with 200 μL RIPA buffer supplemented with antiprotease cocktail.

2.1.6. Incubate on ice 20 min and mix gently every 5 min with a P200 micropipette.

2.1.7. Stock prepared cells at -80℃.

2.1.8. Right before pull-down experiment, thaw prepared cell extract. Centrifuge at 17,000×g at 4℃ for 20 min. Use the supernatant as prey by following step 9 in Subheading 2.2.

2.2 Pull-Down Assay

2.2.1. Transfer 120 μL Ni-NTA agarose beads to gravity flow column.

2.2.2. Centrifuge column for 1 min at 1000×g at 4℃. Discard flow-through.

2.2.3. Add 400 μL distilled water to column.

2.2.4. Centrifuge column for 1 min at 1000×g at 4℃. Discard flow-through.

2.2.5. Mix carefully 50 μg His-tagged protein (bait) with 400 μL equilibrium buffer and load onto column.

2.2.6. Incubate 1 h with agitation at 4℃ and 10 min on ice without agitation.

2.2.7. Centrifuge column for 1 min at 1000×g at ℃ and keep flow-through.

2.2.8. Load flow-through to column, and centrifuge column for 1 min at 1000×g at 4℃. Keep flow-through at 4 °C for analysis.

2.2.9. Mix 200 μL cell extract with 200 μL equilibrium buffer and load onto column.

2.2.10. Incubate 1 h at 4℃ under agitation then 10 min on ice without agitation.

2.2.11. Centrifuge column for 1 min at 1000×g at 4℃. Keep flowthrough for analysis.

2.2.12. Wash column by adding to column 400 μL equilibrium buffer.

2.2.13. Centrifuge column for 1 min at 1000×g at 4℃. Discard flow-through.

2.2.14. Wash column by adding to column 400 μL equilibrium buffer containing 50 mM imidazole. Keep the first washing for analysis.

2.2.15. Centrifuge column for 1 min at 1000×g at 4℃. Discard flow-through.

2.2.16. Repeat steps 14 and 15 three times and go to step 17. Keep last washing fraction at 4℃ for analysis.

2.2.17. Elute by loading 80 μL elution buffer to column and incubate 10 min at 4℃).

2.2.18. Centrifuge column for 1 min at 1000×g at 4℃ and keep eluted fraction.

2.2.19. Repeat steps 17 and 18 with eluted fraction. Keep eluted fraction at 4℃ for analysis.

3. SDS-PAGE and Analysis of Protein Fractions

3.1. To 15 μL protein fraction add 5 μL Laemmli lysis buffer, 4×concentrate. Heat for 3 min at 100℃and centrifuge 30s using a microcentrifuge to bring down condensate.

3.2. Load 10 μL protein fraction and 5 μL protein ladder on SDS-polyacrylamide gel.

3.3. Electrophorese proteins in running buffer at 100 V for 15 min then 180 V until dye front has reached bottom of gel.

3.4. Identify interacting proteins by immunodetection or blue coomassie coloration.

1. We prefer not to use the solutions after 6 months of storage.

2. A different buffer, such as HEPES, MES, or phosphate buffers, may be required for your specific protein–protein interaction. Additionally, different pH values may be tested as these are specific and dependent on the interaction between proteins.

3. The pull-down experiments work better with fresh equilibrium and elution buffers.

4. Make an aliquot of 1 mL before−20℃ storage. This will prevent the degradation caused by repeated thawing.

5. Make an aliquot of 500μL before −20℃ storage. The used Laemmli lysis buffer can be kept at 4℃ for 1 month.

6. As negative control, prepare a cell lysate without expressing bait protein (negative cell lysate). This will eliminate false positives resulting from nonspecific interactions of cell lysate proteins with the Ni-NTA agarose beads.

7. Whole-cell lysate instead of the supernatant fraction can also be used to test whether the prey protein of interest localizes in the pellet fraction.

8. Try to work mostly on ice or at 4℃to prevent the degradation or the denaturation of the proteins.

9. The incubation time can be increased from several hours to overnight at 4℃ under agitation depending on the strength of the interaction between bait and prey proteins.