Product Information

Product Description

GSTPure Glutathione Agarose Resin is a conventional GST-tagged protein purification resin. Its structure is based on highly cross-linked 4% agarose gel as the matrix. It is made by covalently binding reduced glutathione through a 12-atom spacer arm using a chemical method. It has high physical and chemical properties, can withstand high pressure, and can achieve protein purification at a relatively high flow rate. It is suitable for large-scale industrial protein purification.

In addition, this product has good specificity and high cost-effectiveness, and can purify glutathione transferase and glutathione-dependent proteins in various expression systems in one step.

Product Nature

Transportation and storage methods

Transport with ice packs. Store at 4℃, valid for 2 years.

Reagents to prepare

1. Before use, water and buffer should be sterilized by filtration using a 0.22 μm or 0.45 μm filter membrane.

2. Binding/washing buffer : PBS, pH 7.4 (140 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 1.8 mM KH ₂ PO ₄ , pH 7.4)

3. Elution buffer : 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0

Note: 1-10 mM DTT can be added to the binding/wash buffer or elution buffer.

 

How to use

Note: It is best to filter the sample with a 0.22 μm or 0.45 μm filter membrane before loading to reduce impurities and prevent column clogging.

1. Filling of fillers (applicable to filling of various medium-pressure chromatography columns)

1) Rinse the sieve plate and joints at the bottom of the chromatography column with deionized water, ensure that there are no bubbles on the sieve plate at the bottom of the column, close the outlet at the bottom of the column, and leave 1-2 cm of deionized water at the bottom of the column.

2) Suspend the resin and carefully pour the slurry into the chromatography column continuously. Use a glass rod to pour the slurry along the column wall to reduce the generation of bubbles.

3) If a reservoir is used, immediately fill the column and reservoir with water, place the injection distributor on the surface of the slurry, and connect it to the pump, avoiding the formation of bubbles in the distributor or injection tube.

4) Open the outlet at the bottom of the column, start the pump, and run it at the set flow rate. Initially, the buffer should flow slowly through the column, and then slowly increase to the final flow rate. This can avoid the impact of hydraulic pressure on the formed column bed and avoid uneven formation of the column bed. If the recommended pressure or flow rate cannot be achieved, the maximum flow rate of the pump used can be used, which can also achieve a good filling effect.

Note: In the subsequent chromatography procedure, do not exceed 75% of the maximum column packing flow rate. When the column bed height is stable, add at least 3 times the column bed volume of deionized water at the final column packing flow rate. Mark the column bed height.

5) Turn off the pump and close the chromatography column outlet.

6) If using a reservoir, remove the reservoir and place the dispenser in the column.

7) Push the distributor toward the column to the marked column bed height. Allow the column liquid to enter the distributor and lock the distributor joint.

8) Connect the packed column to the pump or chromatography system and start equilibration. If necessary, readjust the dispenser.

2. Sample purification 1) Balance: Use 5 times column volume of binding buffer to balance the column so that the filler is in the same buffer system as the target protein to protect the protein.

2) Loading: After the resin is equilibrated, add the protein sample to allow it to fully contact with the resin to increase the recovery rate of the target protein and collect the effluent.

3) Washing: Use 10-15 column volumes of washing buffer to remove non-specifically adsorbed impurities and collect the washing solution.

4) Elution: Use 5-10 times column volume of elution buffer and collect the eluate, i.e. the target protein solution.

5) Cleaning and storage: Use 3 column volumes of binding buffer and 5 column volumes of deionized water to equilibrate the filler, and finally equilibrate with 5 column volumes of 20% ethanol. Then store in an equal volume of 20% ethanol and store at 4°C to prevent the filler from being contaminated by bacteria.

3. SDS-PAGE detection

The samples obtained during the purification process (including original samples, effluent fractions, washed impurities and eluted fractions, etc.) were detected using SDS-PAGE to determine the purification effect.

4. Filler cleaning

GST-tagged protein purification products can be reused without regeneration, but with the increase of non-specific binding proteins and protein aggregation, the flow rate and binding capacity performance will decrease, and the filler needs to be cleaned.

1 ) Remove some sediment or deformed materials

Wash with 2 column volumes of 6 M guanidine hydrochloride solution and immediately wash with 5 column volumes of PBS, pH 7.4.

2 ) Remove some non-specific adsorbed substances caused by hydrophobic adsorption

Wash with 3-4 column volumes of 70% ethanol or 2 column volumes of 1% Triton X-100, followed immediately by 5 column volumes of PBS, pH 7.4.

Precautions

1) Do not freeze this product.

2) Before using the GST filler, be sure to invert it several times to mix the agarose beads evenly.

3) During all operations, samples need to be operated at 4°C or on ice.

4) For your safety and health, please wear a lab coat and disposable gloves when operating.

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