Purification of GST-tagged proteins using PureCube Glutathione MagBeads
This protocol describes the generation of a cleared lysate from an E. coli cell pellet and the subsequent purification of GST-tagged proteins under native conditions using PureCube Glutathione MagBeads. Reagent amounts given apply to 10 mL IPTG-induced bacterial culture of a well-expressed protein (approximately 10–50 mg/L). In this protocol, cell lysis is done using lysozyme because it is an inexpensive and efficient method for cells that have been frozen. However, lysis methods using detergents (e.g., CHAPS) can also be used. The GST-tagged target protein is purified from cleared lysate under native conditions in a bind-wash-elute procedure.
Buffer compositions - Native protein purification protocol
Lysis Buffer, 100 mL
| |
| Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for stock | Stock needed for buffer |
|---|
| Tris-HCl, pH 7.4 |
125 mM |
121.14 |
0.5 M |
30.29 g/500 mL |
25 mL |
| NaCl |
150 mM |
58.44 |
5 M |
146.1 g/500 mL |
3 mL |
| DTT |
1 mM |
154.25 |
1 M |
14.6 g/100 mL |
200 µL |
| EDTA |
1 mM |
292.24 |
0.5 M |
1.54 g/10 mL |
100 µL |
| Lysozyme |
1 mg/mL |
- |
100 mg/mL |
1 g /10 mL |
1 mL |
| Triton X-100 |
1% (v/v) |
- |
100%(v/v) |
- |
1 mL |
| Protease inhibitor |
1x |
- |
- |
- |
2 tablets |
| Instructions: Prepare a 0.5 M Tris-HCl stock by dissolving Tris base in 400 mL deionized water, adding HCl to a pH of 7.4, and adding water to a final volume of 500 mL. Lysis buffer should always be prepared fresh. |
Wash Buffer, 100 mL
| |
| Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for stock | Stock needed for buffer |
|---|
| Tris-HCl, pH 7.4 |
125 mM |
121.14 |
0.5 M |
30.29 g/500 mL |
25 mL |
| NaCl |
150 mM |
58.44 |
5 M |
146.1 g/500 mL |
3 mL |
| DTT |
1 mM |
154.25 |
1 M |
1.54 g/10 mL |
100 µL |
| EDTA |
1 mM |
292.24 |
0.5 M |
14.6 g/100 mL |
200 µL |
| Instructions: Optimal buffer conditions may vary depending on the protein of interest. Proteins may require addition of protease inhibitor cocktail, EDTA, 1-5 mM DTT, 1% BSA, or detergents such as 0.5-1% Igepal CA-630 (Nonindet P-40) or 0.5-1% Tween-20. |
ATP Buffer (optional), 10 mL
| |
| Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for stock | Stock needed for buffer |
|---|
| Tris-HCl, pH 7.4 |
50 mM |
121.14 |
0.5 M |
30.29 g/500 mL |
1 mL |
| ATP |
2 mM/td> |
551.14 |
100 mM |
551 mg/1 mL |
200 µL |
| MgSO4 |
10 mM |
120.37 |
1 M |
1.24 g/ 1 mL |
100 µL |
| Instructions: Add water to 10 mL. Always prepare fresh. |
Elution Buffer, 10 mL
| |
| Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for stock | Stock needed for buffer |
|---|
| Tris-HCl, pH 7.4 |
125 mM |
121.14 |
0.5 M |
30.29 g/500 mL |
2.5 mL |
| NaCl |
150 mM |
58.44 |
5 M |
146.1 g/ 500 mL |
300 µL |
| Triton X-100 |
0.1% (v/v) |
- |
100% (v/v) |
- |
10 µL |
| Reduced glutathione |
50 mM |
307.32 |
- |
- |
154 mg |
| DTT |
1 mM |
154.25 |
1 M |
1.54 g/10 mL |
10 µL |
| Instructions: Dissolve in 8 mL water, stir until the reduced glutathione is completely dissolved. Depending on the protein‘s requirements, set the pH to 7.4-8.0 using NaOH, then add water to 10 mL. Always prepare fresh. |
5X SDS Page Buffer, 10 mL
| |
| Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for stock | Stock needed for buffer |
|---|
| Tris-HCl, pH 6.8–7.0 |
300 mM |
121,14 |
1 M |
121,14 g/ 1 L |
3 mL |
| Glycerol |
50% (v/v) |
- |
100% (v/v) |
- |
5 mL |
| SDS |
5% (w/v)) |
- |
- |
- |
0.5 g |
| Bromophenol blue |
0.05% (w/v) |
- |
4% |
- |
125 μL |
| DTT |
250 mM |
154.25 |
1 M |
1.54 g/ 10 mL |
125 μL/aliquot |
| Instructions: Make sure to prepare a 1 M Tris-HCl stock by dissolving Tris base in 500 mL deionized water, adding HCl to a pH of 6.8–7.0, and adding water to a final volume of 1 L. For the SDS-PAGE Buffer, mix all components listed except DTT and add water to a total of 10 mL. Freeze 20 aliquots (375 µL each) at –20˚C. Before use, add DTT to the needed single aliquots. |
Table 1. Magnetic bead suspension volumes suitable for given protein expression levels
| |
| Protein expression level | Amount of GST-tagged protein per 1 mL culture | Amount GSTtagged protein per 10 mL* culture | Volume 25% magnetic bead suspension per 10 mL culture | Minimum elution volume per 10 mL culture |
|---|
| >0.5 mg/L |
>0.5 μg |
>5 μg |
>2 μL* |
>25 μL |
| 1 mg/L |
1 μg |
10 μg |
4 μL* |
25 μL |
| 5 mg/L |
5 μg |
50 μg |
20 μL |
50 μL |
| 10 mg/L |
10 μg |
100 μg |
40 μL |
100 μL |
| 50 mg/L |
50 μg |
500 μg |
200 µL |
500 μL |
| *Note: For easier handling, dilute the magnetic bead suspension in PBS buffer pH 7.4 before use. |
| |
|
| A. | Protein purification protocol: |
|---|
| |
- Thaw the E. coli cell pellet on ice. Optional: Freezing the cell pellet at -20 °C for 30 min prior to incubation at room temperature improves lysis by lysozyme.
- Resuspend the cell pellet in 1 mL Lysis Buffer. Add 30 U Benzonase® (3 units/mL bacterial culture) to the lysate to reduce viscosity caused by genomic DNA.
- Incubate for 30 min on ice, if necessary for protein stability. Otherwise, incubating at room temperature (20-25 °C) may be more efficient.
- Centrifuge the lysate for 30 min at 10,000xg and 2-8 °C. Collect the supernatant. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
- Resuspend the PureCube Glutathione MagBeads by vortexing. Transfer 40 μL of the 25 % magnetic beads suspension into a conical microcentrifuge tube. Note: Depending on the protein expression rate, the quantity of magnetic bead suspension can be adjusted from 2-200 μL.
- Add 500 μL Lysis Buffer to the Glutathione MagBeads and mix by vortexing. Place the tube on a magnetic microtube stand until the beads are separated and discard the supernatant.
- Pipet 1 mL of the cleared lysate onto the equilibrated MagBeads, and incubate the mixture at 4 °C for 1 h on an end-over-end shaker.
- Place the tube on the magnetic microtube stand until the beads separate and remove the supernatant.
- Remove the tube from the magnet. Add 500 μL Wash Buffer and mix by vortexing. Place the tube again on the magnetic microtube stand and allow the beads to separate. Remove the supernatant.
- Repeat step 10 twice. 12: Optional: To remove contaminants such as chaperones, perform an additional wash step with ATP buffer.
- Elute the GST-tagged protein using 100 μL Elution buffer. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 μL.
- Repeat step 12xxx Collect each elution fraction in a separate tube.
- Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate the sample at 46 °C for 30 min in preparation for SDS-PAGE analysis.
- Optional: Perform Western Blot experiment using GST Antibody.
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