GST, or Glutathione S-Transferase, is expressed as a fusion with the target protein to generate a GST-tagged recombinant protein, also known as a GST-tagged protein or GST fusion protein. GST-tagged proteins bind specifically to GST Affinity Resin, while other proteins do not. Bound GST-tagged proteins can be eluted with excess reduced glutathione (GSH), enabling the separation and purification of the target protein. Since purification is performed under mild, native conditions, the target protein retains its biological activity, making it suitable for structural and functional studies, antibody preparation, protein‑protein interaction assays, and protein‑nucleic acid interaction studies. This kit supports purification of GST‑tagged proteins expressed in Escherichia coli, mammalian cells, insect cells, and baculovirus systems. GST fusion proteins often show high solubility in E. coli and help maintain the native activity of the target protein. Many eukaryotic proteins expressed in E. coli form inclusion bodies, but a significant portion can be expressed in soluble form when fused to GST, facilitating subsequent purification. If a site‑specific protease cleavage site, such as the recognition sequence for TEV Protease (rp155927), is inserted between the GST tag and the target protein, the GST tag can be removed using the corresponding protease. Fusing GST to the N‑terminus of the target protein generally preserves GST enzymatic activity and simplifies purification using GST Affinity Resin.
Key component: GST Affinity Resin
Matrix: 4% cross‑linked agarose
Ligand: reduced glutathione, covalently coupled via a 12‑atom spacer arm
Binding capacity: >20 mg GST fusion protein per mL settled resin
Particle size: 45-165 μm
Maximum operating pressure: 0.1 MPa (1 bar)
The resin is supplied in 1×PBS containing 20% ethanol. A 10 mL slurry consists of 5 mL settled resin and 5 mL storage solution. The resin must be fully resuspended before pipetting. Actual binding capacity depends on the molecular weight of the target GST‑tagged protein: higher molecular weight generally results in higher binding capacity. This kit is sufficient for purification of up to 10 GST‑tagged proteins, with a typical yield of approximately 10 mg per protein.
Product Features
Rapid and Efficient: The kit uses high‑performance GST affinity resin with strong binding efficiency, shortening operation time and effectively preventing degradation or denaturation of the target protein during prolonged handling, fully preserving protein activity.
Convenient: All required reagents and empty gravity chromatography columns are provided, greatly simplifying purification of GST‑tagged proteins.
High Binding Capacity: 20 mg GST fusion protein can be bound per mL of settled resin.
High Specificity: Enables one‑step purification of glutathione S‑transferases, glutathione‑dependent proteins, and GST recombinant derivatives from various expression systems.
Instruction for use
1 Sample Preparation
1.1 Soluble Protein Expressed in Bacteria or Yeast
1.1.1 Pick a single colony into medium containing the appropriate antibiotic. Induce expression with inducer at the recommended concentration and duration according to the vector instructions.
1.1.2 After expression, transfer the culture to a centrifuge bottle and centrifuge at 7000 rpm (7500 ×g) for 15 min to harvest cells. Resuspend the cell pellet in Native Lysis Buffer at a ratio of 1:10 (w/v). Optionally add PMSF to a final concentration of 1 mM or other protease inhibitors if the protein is prone to degradation, provided they do not interfere with binding to the resin. Lysozyme may be added as needed (not required if the host strain contains pLysS or pLysE).
1.1.3 Fully resuspend the cell pellet. If the cell suspension is concentrated, add 10 μg/mL RNase A and 5 μg/mL DNase I. Mix well, keep on ice, and lyse by sonication in an ice-water bath until the lysate becomes clear.
1.1.4 Transfer the lysate to a centrifuge tube and centrifuge at 10000 rpm (15000 ×g) at 4 °C for 20-30 min. Collect the supernatant, keep on ice, and proceed to purification.
1.2 Soluble Protein Expressed in Yeast, Insect, or Mammalian Cells
Transfer the cell culture to a centrifuge bottle and centrifuge at 5000 rpm (3800 ×g) for 10 min. Collect the supernatant. If the supernatant contains no EDTA, histidine, or reducing agents, it may be loaded directly. If EDTA, histidine, or reducing agents are present, dialyze against wash buffer before loading. For large volumes, concentrate by ammonium sulfate precipitation and dialyze against wash buffer before loading.
2 Buffer Preparation
2.1 10×GSH Stock Solution:
Dissolve the 184 mg GSH provided in the kit in 6 mL Elution Buffer and mix thoroughly.
Store the prepared 10×GSH solution at -20 °C, stable for at least 1 year.
2.2 1×GSH Elution Buffer:
Mix Elution Buffer and 10×GSH solution at a 9:1 ratio (e.g., 9 mL Elution Buffer + 1 mL 10×GSH). Since GSH is easily oxidized and inactivated, prepare fresh before use. The prepared 1×GSH Elution Buffer may be stored at 4 °C and used within 2 weeks.
3 Sample Purification
Before loading, samples are recommended to be centrifuged or filtered through a 0.22 μm or 0.45 μm filter to reduce impurities, improve purification efficiency, and prevent column clogging. Buffers are also recommended to be filtered through a 0.22 μm or 0.45 μm filter before use.
3.1 Gravity Column Purification
3.1.1 Column Packing
3.1.1.1 Take an empty gravity column, insert the bottom frit, rinse with purified water, and close the bottom outlet.
3.1.1.2 Mix the GST Affinity Resin thoroughly and pipette the slurry into the gravity column. The settled resin volume is approximately half the slurry volume (e.g., 1 mL slurry yields ~0.5 mL resin, with a maximum capacity of ~10 mg). Open the outlet to drain the storage solution.
3.1.1.3 Wash the resin with purified water. Drain completely and close the outlet.
3.1.1.4 Insert the top frit, ensuring no air gap between frit and resin and that the frit is level.
3.1.1.5 The packed column may be equilibrated immediately with wash buffer. If not used immediately, add storage solution and store at 4 °C.
3.1.2 Equilibration
Equilibrate the column with 2-5 CV Lysis Buffer (CV = settled resin volume; this definition applies throughout the protocol). Repeat 2-3 times.
3.1.3 Sample Loading
Apply the sample to the equilibrated column. Allow the sample to flow slowly to ensure sufficient binding. Collect the flow-through; reloading may increase binding efficiency. Keep samples on ice if the target protein is unstable.
3.1.4 Washing
Wash with 5-10 CV Lysis Buffer to remove non‑specifically bound proteins. Collect the wash fraction. During washing and subsequent elution, protein concentration in each wash and elution fraction can be rapidly monitored using the Bradford method to adjust the number of washing and elution steps as needed.
3.1.5 Elution
Elute the target protein with 5-10 CV 1×GSH Elution Buffer. Collect fractions every 1-5 CV and analyze individually to ensure complete elution and obtain highly pure, concentrated protein. After elution, wash with 3 CV equilibration buffer, 5 CV purified water, and 2 CV storage solution. Store the resin at 2-8 °C.
3.2 Batch Purification
3.2.1 Resin Preparation
Transfer an appropriate volume of GST Affinity Resin into a centrifuge tube. Centrifuge at 1000 rpm for 1 min and carefully remove the supernatant (use low speed to avoid damaging the resin). Alternatively, drain the storage solution in a gravity colum.
3.2.2 Resin Equilibration
Add 5 CV Lysis Buffer, mix gently by inversion, centrifuge at 1000 rpm for 1 min, and carefully remove the supernatant. Repeat at least twice. For gravity columns, wash and drain by gravity.
3.2.3 Binding
Add the prepared sample to the equilibrated resin. Seal the tube or column and mix by rotation. Incubate at room temperature for 30 min-2 h or at 2-8 °C for 2-4 h (longer if needed). Keep at 2-8 °C if the protein is unstable. Centrifuge at 1000 rpm for 1 min and retain the supernatant for analysis.
3.2.4 Washing
Wash with 5 CV Lysis Buffer, mix gently, centrifuge at 1000 rpm for 1 min, and retain the supernatant. Repeat 3-5 times. For gravity columns, wash and drain by gravity.
3.2.5 Elution
Elute with 3-5 CV 1×GSH Elution Buffer. Incubate at room temperature for 5 min. Centrifuge at 1000 rpm for 1 min or collect by gravity flow. Repeat 2-3 times. After elution, wash with 3 CV equilibration buffer, 5 CV purified water, and 2 CV storage solution. Store the resin at 2-8 °C.
4 Sample Detection and Resin Cleaning
4.1 SDS-PAGE
Analyze samples from the purification process (starting material, flow-through, wash, and elution fractions) by SDS-PAGE to evaluate purification efficiency.
4.2 Resin Cleaning
After repeated use, cleaning is required (frequency depends on sample contaminants).
General cleaning: Wash with 2 CV 6 M guanidine hydrochloride, then immediately with 5 CV purified water, then 2 CV storage solution. Store at 2-8 °C.
Removal of hydrophobic contaminants: Wash with 3-4 CV 70% ethanol or 2 CV 1% Triton X‑100, then immediately with 5 CV purified water, then 2 CV storage solution. Store at 2-8 °C.
Matters needing attention
1. Do not freeze the GST Affinity Resin.
2. GST Affinity Resin is supplied in 1×PBS containing 20% ethanol, with a 1:1 ratio of settled resin to storage solution.
3. The resin must be thoroughly resuspended by repeated inversion before use to ensure uniform agarose bead suspension.
4. This product is for research use only.
5. For your safety and health, wear a lab coat and disposable gloves during all experiments.
