GST fusion protein pulldown assays.

GST Fusion Protein Purification and Pull-down Protocol Protocol:

Day 1:

1. Inoculate about 5 ul of Glycerol Stock (stored at -80oC) of E. coli expressing the GST fusion protein of interest (and GST as control) in 10 ml LB media with the appropriate antibiotic. Grow overnight with shaking at 37°C.

Day 2:

1. Dilute the overnight grown culture in 100 ml of LB media with antibiotic. Grow at 37°C with shaking to an optical density at 600 nm of between 0.6-0.8.
2. Add IPTG to a final concentration 1 mM.
3. Grow at 37°C with shaking for 3-4 hours.
4. Harvest bacteria in 50 ml Falcon conical tubes. Spin at 3500 RPM for 20 minutes at 4 degrees in a benchtop centrifuge. Discard the supernatant. At this point, the pellets can be frozen for later use, or can be used immediately.
5. Resuspend the pellet in 10 ml ice cold lysis buffer (using a plastic pipette) and rock at 4oC (in a cold room) for 20 min.
6. Sonicate in 15 ml falcon tubes at 30% power three times for 20 sec each, keeping on ice in between sonication cycles to allow the foam to settle down (2-5 minute). (Fisher Scientific Sonic Dismembrator Model 500)
7. Centrifuge the bacterial lysate for 30 min at 20K RPM at 4°C (Thermo Scientific F21- 8x50y rotor)
8. Transfer the supernatant into a new tube and add 100 ml of washed 20% Glutathione- Sepharose beads.
9. Rock at 4°C for 2-3 hours
10. Spin down the beads at 1500 RPM for 2 min at 4°C. Save the supernatant.
11. Wash the beads with bead wash buffer six times at 1500 RPM for 2 min at 4 degrees.
12. Resuspend the beads in 100 ul of bead wash buffer.
13. Determine protein concentration of bead suspension using the BCA protein estimation kit. Run 1-10 mg samples on an SDS-PAGE gel alongside pre-stained or unstained protein markers followed by Coomassie staining to verify the size and intactness of the fusion proteins being analyzed.
14. Store the beads at 4°C for use over up to 2 weeks.

Pull down assay:

1. Lyse the cells using cell lysis buffer. Determine protein concentration (BCA method).
2. Add Glutathione-Sepharose beads coated with 20ug of GST fusion protein (or GST as a control) to 500ug - 1mg of cell lysate. Use uncoated glutathione-Sepharose beads as an additional control.
3. Rock at 4°C for 3 hours.
4. Wash the beads with cell lysis buffer six times at 1500 RPM for 2 min at 4°C.
5. Finally resuspend the beads in ~50 ml Laemmli Sample Buffer.
6. Resolve by SDS-PAGE and subject to Western blotting as indicated.

Preparation of Glutathione-Sepharose beads:

1. Spin down the needed amount of Glutathione-Sepharose bead slurry (in ethanol preservative) for 2 minutes at 4°C in a benchtop centrifuge or 15 sec in a microfuge.
2. Resuspend in at least 10 volumes of bead wash buffer and spin as before. Repeat washing a total of 3 times. 3. Resuspend the beads in blocking buffer at a final concentration of 20% beads and rock for an hour at 4°C. Beads can be stored as such for further use (typically, less than 2 weeks).
3. For experiments, pipette the beads up and down to make a uniform suspension and take the required amount in a fresh 15-ml conical or Eppendorf tube. Wash the beads with bead wash buffer and resuspend in fresh lysis buffer at 20% for use in the experiment.
    

Buffers:

Bacterial Lysis Buffer:

50 mM Tris, pH 7.5 (Fisher Chemicals, Catalog # BP152-5)

150 mM NaCl (Fisher Chemicals, Catalog # S271-10)

0.5% Tx-100 (Sigma-Aldrich Catalog # 93418)

1 mM PMSF (Thermo-Scientific Catalog # 36978)
 

Tris-buffered Saline-Triton (TBS-T) buffer:

50 mM Tris, pH 7.5

150 mM NaCl

0.1% Tx-100
 

Bead Wash Buffer:

TBS-T plus:

0.02% Sodium azide (Fisher Scientific CAS # 26628-22-8)
 

Bead Blocking Buffer:

TBS-T plus:

0.1% BSA (Sigma-Aldrich Catalog # A7906-100G)

0.02% Sodium azide
 

Cell Lysis Buffer:

50 mM Tris pH 7.5

150 mM NaCl

0.5% Tx-100

10 mM Sodium fluoride (Fisher Chemicals Catalog # S299-500)

1 mM Sodium orthovanadate (Sigma-Aldrich Catalog # S6508-50G)

0.5 mM PMSF (Thermo-Scientific, Catalog # 36978)
 

Other Reagents:

LB Broth Miller (Fisher BioReagents Catalog # BP9723-2) IPTG (Research Products International Catalog # I56000)

Glutathione Sepharose 4B (Global Life Sciences Catalog # 17075601)

How to cite: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

1. Mushtaq I, Band V, Band H (2021). GST Fusion Protein Purification and Pull-down Protocol. Bio-protocol. Xxxxx.
2. Tom EC, Mushtaq I, Mohapatra BC, Luan H, Bhat AM, Zutshi N, Chakraborty S, Islam N, Arya P, Bielecki TA, Iseka FM, Bhattacharyya S, Cypher LR, Goetz BT, Negi SK, Storck MD, Rana S, Barnekow A, Singh PK, Ying G, Guda C, Natarajan A, Band V, Band H. EHD1 and RUSC2 Control Basal Epidermal Growth Factor Receptor Cell Surface Expression and Recycling. Mol Cell Biol. 2020 Mar 16;40(7):e00434-19. doi: 10.1128/MCB.00434-19.

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