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How does the GST-tag Affinity Media Work?

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  • Time of issue:2022-07-20 15:38
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(Summary description)

How does the GST-tag Affinity Media Work?

(Summary description)

  • Categories:News
  • Author:
  • Origin:
  • Time of issue:2022-07-20 15:38
  • Views:
Information

How does the GST-tag Affinity Media Work?

 

GST-tag Affinity media

 

Affinity chromatography media has high selectivity for ligands, and it is an ideal purification technique to introduce affinity chromatography in the stage of sample capture and crude purification. Therefore, in the process of protein expression using a prokaryotic expression system, a specific ligand tag is usually added to the vector for fusion expression with the target protein. At present, the commonly used affinity chromatography includes His tag affinity chromatography and GST-tag affinity chromatography.

 

GST-tag affinity chromatography utilizes the principle that GST fusion protein and immobilized glutathione (GSH) are covalently bound through sulfur bonds and are purified by GSH exchange and elution. Glutathione is specifically coupled to the agarose medium through the SH group and the ethylene oxide group on the agarose medium through epoxy activation, and then utilizes the specific interaction between the enzyme and the substrate between it and the GST-tag the force allows the GST-tagged fusion protein to bind to GSH on the gel, thereby separating the tagged protein from other proteins. In addition, the introduction of the GST-tag can make the target protein (exogenous protein) fold better, increase its solubility and stability, and better exert its biological activity and antigenicity.

 

Nanotein GST Starose 4 Fast Flow

 

Specifications:

 

Different specifications of GST Starose 4 Fast Flow prepacked products are available, including 1 mL prepacked columns, 5 mL prepacked columns, and 10-110 mL XPXK prepacked columns.

 

Features:

 

● Expression of recombinant proteins or domains fused to GST using pGEX vectors can be easily purified by GST Starose 4 Fast Flow affinity chromatography under mild conditions.

 

● Average particle size (90 μm), the pressure resistance of 0.3 MPa, and maximum flow rate of 600 cm/h. It can be expanded from laboratory-level process exploration to industrial-scale production.

 

● The pH stability range (3~12) is large, which can meet the working and cleaning conditions of different conditions.

 

Application Cases:

 

The relevant protein purification was performed using the GST Starose 4 Fast Flow, and the following results were obtained:

 

● Good purification effect for GST-tag fusion proteins (GST-tag Pro-1, -2, -3, -4) from different expression levels and sizes.

 

● High stability for multiple replicates of the same sample (GST-tag Pro-4).

 

● Easily amplify and purify the same sample (GST-tag Pro-4) from 1 mL to 20 mL.

 

Materials and Methods

 

Case 1: Purification of Recombinant GST-tagged Fusion Protein

Figure 1. GST-tag Pro-1 gradient elution chromatogram and SDS-PAGE identification

 

Analysis: After the sample was equilibrated, linear gradient elution was performed to obtain a high-purity GST-tagged fusion protein (about 30 kDa) without an obvious target protein flow-through.

 

Case 2 Purification of recombinant GST-tagged fusion protein

Figure 2. GST-tag Pro-2 one-step elution chromatogram and SDS-PAGE identification

 

Analysis: One-step elution was performed after the sample was equilibrated, and a high-purity GST-tagged fusion protein (about 80 kDa) was obtained, and there was no obvious target protein flow-through.

 

Case 3 Purification of recombinant GST-tagged fusion protein

 

Figure 3. GST one-step elution chromatogram and SDS-PAGE identification

 

Analysis: Gradient elution was performed after the sample was equilibrated to obtain a GST-tagged fusion protein with high purity (about 50 kDa), and there was no obvious target protein flow-through.

 

Case 4 6 repeated purification stability verification

Figure 4. GST one-step elution process stability validation SDS-PAGE identification

 

Analysis: The same sample was used for 6 repeated purifications of the same process, during which the GST medium was not cleaned or regenerated, and the purification was repeated with excellent reproducibility of the purity and recovery of the elution results.

 

Case 5 1 mL to 20 mL column volume scale-up experiment

Figure 5. Scale-up of the GST purification process from a 1 mL prepacked column (left) to a 16/10 20 mL column (right)

 

Figure 6. Scale-up SDS-PAGE identification of GST gradient elution process

 

Analysis: The purification scale of GST-tag Pro-4 protein was enlarged from a 1 mL prepacked column to a 20 mL chromatographic column, and the purity of the target protein was 93.8% and 94.5%, and the yields were 96.7% and 97.4%, respectively. Compared with the 1 mL prepacked column, the purification of the protein using a 20 mL chromatographic column showed a slight increase in purity and yield, indicating that GST Starose 4 Fast Flow can realize the purification of GST tags without affecting the purity and yield. Scale-up of protein purification processes.

 

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