Pull Down Assay (PDA)

         Detection of functional complex formed by direct physical interaction between protein molecules forms the basis of understanding various signalling pathways as well as cross talk between different pathways. GST pull-down assay  is an important technique useful for both confirming the protein-protein interaction predicted by other research techniques (e.g., co-immunoprecipitation, yeast two hybrid) as well as for screening of novel /unknown protein        interactions.

     A. Reagents and Equipment

         Reagents - GST pull down assay

  • 50% slurry of Glutathione Sepharose 4B
  • Protein extraction buffer: 1mM DTT, 1 mM PMSF, 1 mM EDTA (pH 8.0) in 1X PBS pH 7.4
  • 1X PBS pH 7.4
  • IPTG (isopropyl-β-D-thiogalactoside)
  • LB broth
  • Lysozyme (100 mg/ml stock in water .Storage at 20 oC)
  • DNase (10 µg/ml stock solution in 50 mM HEPES, 250 mM KCl, 1 M DTT and 50% glycerol)
  • Triton X 100 (0.2%)
  • Glutathione elution buffer: 20 mM reduced glutathione in 50 mM Tris-HCl (pH 8.0)

         Reagents - In-gel protein digestion for mass spectrometry

  • Distaining and wash solution: 50 mM ammonium bicarbonate in 50% Acetonitrile
  • Digestion Buffer: 50 mM ammonium bicarbonate in water
  • Reducing Buffer: 10 mM Dithiothreitol (DTT) in 50 mM ammonium bicarbonate (freshly prepared)
  • Alkylating Buffer : 55 mM iodoacetamide in 50 mM ammonium bicarbonate (freshly prepared )
  • Trypsin solution: To perform 10 reactions, 5 µl of trypsin (0.2 µg/ µl) is taken and to it, 95 µl of digestion buffer is added. Mix it properly and then use.
  • Formic acid
  • Trifluoroacetic acid

         Equipment

  • 4 °C microcentrifuge.
  • Rotatory shaker incubator
  • Rocker shaker
  • Laminar flow hood 
  • Micro centrifuge and Falcon tubes

     

     B. GST pull down assay 

         Preparation of bacterial cell lysate with expressed GST/ GST fusion protein 

  1. Inoculate 5ml of LB broth with ampicillin (100 μg/ ml) in a culture vial with a single colony of BL21 strain of E.coli (transformed with empty pGEX4T-2 or protein of interest cloned in pGEX4T-2) and incubate overnight at 37 °C with shaking at 200 rpm.
  2. Inoculate 1% of overnight grown culture into 100 ml of LB ampicillin (100 μg/ ml) in a conical flask (capacity of 500 ml) and incubate at 37 °C with shaking until O. D.  600 of 0.6 is achieved.
  3. Induce the culture with 0.3 mM conc. of IPTG for 4 hours. Harvest the cells by centrifugation at 5500 rpm at 4 °C for 15 min.
  4. Wash the cells twice with 1X PBS. Re-suspend the pellet in protein extraction buffer. Add lysozyme to the concentration of 1 mg/ ml and incubate on ice for 30 min. with occasional mixing in between.
  5. Add Triton X 100 (0.2%) and DNase (5 µg/ ml) to the above mix followed by vigorous shaking. Incubate for 1 hour at 4 °C with gentle shaking to solubilize the fusion protein.
  6. Spin at 10,000 rpm for 10 minutes. Clarify the above supernatant by filtration through 0.4 µm filters.
  7. Note: Cell lysate from BL21 expressing GST (Empty pGEX4T-2) is used as control to compare with the cell lysate expressing GST fusion protein. Both samples are processed simultaneously.

         Immobilization of bait protein (GST/GST fusion protein)

  1. Prepare 50% slurry of Glutathione Sepharose 4B from 75% slurry. Gently shake the vial of Glutathione Sepharose 4B resin to suspend the slurry. Transfer 1.33 ml of slurry into a 15 ml Falcon tube for generating 1 ml bed volume.
  2.  Spin at 2100 rpm for 5 min. Decant the supernatant carefully and add 10 bed volume of ice cold 1X PBS and mix well by inverting. Spin at 2100 rpm for 5 min. followed by decanting of supernatant. Repeat this step two more times.
  3. Finally, add 1.0 ml of 1X PBS to the above slurry to prepare 50% slurry.
  4. Incubate the clarified crude cell lysate expressing GST or GST fusion protein (described above) with 50% slurry for 2-4 hours at 4 °C with gentle shaking
  5. Spin at 500 x g for 5 min. Wash the column three times with 10 bed volume of ice cold 1X PBS.

         Incubation of cell lysate (consisting of interacting prey protein) with immobilized bait protein

  1. Prepare 50% slurry of Glutathione Sepharose 4B from 75% slurry. Gently shake the vial of Glutathione Sepharose 4B resin to suspend the slurry. Transfer 1.33 ml of slurry into a 15 ml Falcon tube for generating 1 ml bed volume.
  2.  Spin at 2100 rpm for 5 min. Decant the supernatant carefully and add 10 bed volume of ice cold 1X PBS and mix well by inverting. Spin at 2100 rpm for 5 min. followed by decanting of supernatant. Repeat this step two more times.
  3. Finally, add 1.0 ml of 1X PBS to the above slurry to prepare 50% slurry.
  4. Incubate the clarified crude cell lysate expressing GST or GST fusion protein (described above) with 50% slurry for 2-4 hours at 4 °C with gentle shaking
  5. Spin at 500 x g for 5 min. Wash the column three times with 10 bed volume of ice cold 1X PBS.

         Separation of interacting protein complex on SDS-PAGE gel

  1.  Load the elutes obtained both from the column containing both immobilized GST and GST fusion proteins into adjacent wells on a 10% SDS PAGE gel with appropriate molecular weight marker.
  2. Stain the gel with either silver stain or Comassie.
  3. Excise the protein bands from the gel for further processing.

     C. In-gel protein digestion for mass spectrometry 

         Excising and de-staining of gel pieces

  1. Inoculate 5ml of LB broth with ampicillin (100 μg/ ml) in a culture vial with a single colony of BL21 strain of E.coli (transformed with empty pGEX4T-2 or protein of interest cloned in pGEX4T-2) and incubate overnight at 37 °C with shaking at 200 rpm.
  2. Wash the gel with sufficient water and place it on a light box to excise the desired bands with a clean scalpel.
  3. Cut the bands in to small pieces (1×1 or 2×2mm) and transfer into a microcentrifuge tube containing water. Tube can be stored at 4 oC until further processing.
  4. Add 200µl of destaining solution to the gel pieces and keep at 37 oC for 30 min. with slow shaking.
  5. Discard distaining solution and repeat the step.
  6. Add 200 µl 100% ACN to the gel pieces and incubate for 10 min with occasional shaking. The gel pieces should now appear white (translucent).
  7. Remove ACN from the tube.

         Reduction and alkylation of gel pieces

  1. Add 100-200 µl of reducing buffer and incubate at 60 oC for 1 hr.
  2. Remove the reducing buffer and allow to cool at room temperature.
  3. Add 200 µl of alkylating buffer for 30 min. in dark at room temperature.
  4.  Remove and discard alkylating buffer
  5.  Wash the sample by adding 200 µl of destaining solution and incubate at 37 ºC for 15 minutes. Then discard destaining solution. Repeat this step once more.

         Shrink gel pieces

  1.  Shrink gel pieces by adding 50-100 µl of acetonitrile. Incubate sample for 15 minutes at room temperature. The gel pieces should now become white (translucent).
  2. Carefully remove acetonitrile and allow gel pieces to air dry for 5-10min.

         Trypsinization of proteins and recovery of fragments

  1. Add 10 µl of the trypsin solution to the tube containing the shrunken gel pieces. Incubate in ice for 10 min and allow gel pieces to swell and absorb the trypsin solution. If 10 µl is not sufficient to cover and fully swell gel pieces, use additional 10 µl.
  2. Add 25 µl of digestion buffer to the tube. Incubate sample at 37 oC for four hours or at 30 oC overnight with shaking.

         Extraction of peptides

  1. Remove digestion mixture and place in a clean tube
  2. To further extract peptides, add 10 µl of 0.5% trifluoroacetic acid or 1% formic acid solution to gel pieces and incubate for 5 minutes. Remove extraction solution and add to digestion mixture. This step also serves to inactivate trypsin.
  3. Centrifuge to remove any gel particles to prevent clogging or damage of columns. Transfer to a fresh Eppendorf and store at 4 oC.