StrepTactin-Pulldown
| Materials | |
|---|---|
| Recombinant protein | Bait protein TwinStrepTagII tagged protein, target proteins (HisSUMO or GST tagged protein of interest) |
| Pull down buffer | 50 mM Tris/Cl pH 8, 150 mM NaCl, 10% Glycerol, 0.05% Tween. Optional: 10 µM ZnCl2 in case of Zinc- finger proteins or 2 mM MgCl2, CaCl2 or MnCl2 in case of metalloproteins) |
| Elution buffer EXT | 50 mM Tris/Cl pH 8, 150 mM NaCl, 50 mM Biotin) store dark and cold |
| StrepTactin Sepharose Beads |
Procedure
Equilibration of beads
- Use 10 µl of StrepTactin-Sepharose beads per sample and transfer into fresh 1.5 ml Eppendorf tube. Add 200 µl pulldown buffer
- Centrifuge at 2700 rcf for 2 min. Remove supernatant
- Equilibrate beads by washing them twice in 200 µl pulldown buffer. Remove supernatant after last washing step
Preparation of bait and target protein
- Centrifuge protein extract prior to pulldown 20 min at 10.000 g to remove any debris or aggregated protein.
- Prepare 130 µl of bait protein (TwinStrepTag-proteinX) solution by taking 2.5 µg of bait protein and adjust with pulldown buffer to a final volume of 130 µl. Take 30 µl of solution, mix with 10 µl 4xSDS and boil for 5 min at 95°C. Refer to as “Input”
- Prepare 130 µl of target protein (eg. HisSUMO-proteinY) solution by taking 5-10 µg of target protein and adjust with pulldown buffer to final volume of 130 µl. Take 30 µl of solution, mix with 10 µl 4xSDS and boil for 5 min at 95°C. Refer to as “Input”
Loading of beads
- Resuspend equilibrated sepharose beads in 100 µl bait protein solution
- Incubate 30 min at room temperature on head to head rotor
- Centrifuge at 2700 rcf for 2 min and remove supernatant (take 30 µl of supernatant and mix with 4xSDS. Boil 5 min at 95°C. Refer to as “unbound” fraction)
- Wash 3x with 100 µl pulldown buffer, mix by harsh shaking and centrifuge (2700 rcf, 2 min)
Pulldown
- Resuspend loaded beads in 100 µl target protein solution
- Incubate 2 h at 4° on head to head rotor
- Centrifuge (2700 rcf, 2min) and remove supernatant (take 30 µl of supernatant and mix with 4xSDS. Boil 5 min at 95°C. Refer to as “unbound” fraction)
- Wash 3x with 100 µl pulldown buffer, mix by harsh shaking and centrifuge (2700 rcf, 2 min)
- Elute under NATIVE conditions by adding 30 µl elution buffer BXT
- Incubate 10 min at room temperature on head to head rotor
- You may repeat this step to increase the yield (but not the concentration)
- Mix eluate with appropriate amount of 4xSDS and boil 5 min at 95°C