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| Isolation and analysis of GST-tagged proteins |
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| Background |
The glutathione S-transferase (GST) enzyme family comprises a long list of cytosolic, mitochondrial, and microsomal proteins which are capable of multiple reactions with a multitude of substrates, e.g. glutathione. Genetic engineers have used glutathione S-transferase to create the so-called GST gene fusion system. Here, the GST tag is used to purify and detect glutathione-binding proteins of interest.
A high-quality monoclonal Anti-GST antibody coupled to µMACS™ MicroBeads is offered by Miltenyi Biotec, allowing specific isolation of recombinant GST fusion proteins on an analytical scale.
For medium- to large-scale purification of GST-tagged proteins, the ProCatch Glutathione Resin is recommended (fig. 1). |
| Isolation and purification of GST-tagged proteins |
The µMACS™ and MultiMACS™ GST Isolation Kit take advantage of the well-established MACS® Technology; thus, allows fast and specific isolation of pure GST-tagged proteins. The advantages of MACS Technology for protein isolation are:
Specificity – high-quality monoclonal antibodies coupled to MACS MicroBeads assure specific isolation of GST-tagged proteins with low background.
Sensitivity – fast reaction kinetics of tiny (50 nm), non-sedimenting MACS MicroBeads allow sensitive isolation even when working with rare proteins.
Speed – less than 2 hours to high-purity GST-tagged proteins!
The µMACS GST Isolation Kit was developed for manual, low-throughput applications with the µMACS Separator.
The procedure can easily be upscaled with the MultiMACS GST Isolation Kits to a semi- or fully-automated, high-throughput processing of up to 96 samples in parallel by utilizing the MultiMACS 96 Separator.
For medium- to large-scale purification of GST-tagged proteins the ProCatch Glutathione Resin is recommended.
Antibody specifications
Target sequence: whole glutathione S-transferase (220-residue polypeptide)
Tag origin: Schistosoma japonicum
Isotype: mouse IgG1 |
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| Figure 1 |
Purification of a 30 kDa glutathione-binding protein with ProCatch Glutathione Resin.
A Marker (lane 1), lysate from cell extract (2), flow-through (3), first eluate (4), and second eluate (5) were separated by SDS-PAGE and Coomassie stained. B Low-pressure column purification protocol. Peak 1 defines non-bound protein fraction (flow-through). Peak 2 corresponds to the eluted glutathione-binding protein. Absorbance was measured at 280 nm. |
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| Details |
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| Products |
| μMACS GST Isolation Kit |
| Tag specification |
- for 40 isolations
Components
- 2 mL μMACS GST MicroBeads
- 2×50 mL Lysis Buffer
- 50 mL Wash Buffer 1
- 5 mL Wash Buffer 2
- 5 mL Elution Buffer
Download datasheet
130-091-370
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| μMACS Anti-GST Starting Kit |
- for 40 isolations
Components
- 1 μMACS GST Isolation Kit
- 1 μMACS Separation Unit
- 1 MACS Multistand
- 2×20 Columns
130-091-493
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| MultiMACS GST Isolation Kit (12×8) |
- for 96 isolations
Components
- 3x2 mL µMACS Anti-GST MicroBeads
- 1x50 mL Equilibration Buffer
- 1x Multi-8 Column Box (12x8)
Download datasheet
130-094-254
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| MultiMACS GST Isolation Kit (4×96) |
- for 384 isolations
Components
- 5x4.6 mL µMACS Anti-GST MicroBeads
- 1x50 mL Equilibration Buffer
- 1x Multi-96 Column Box (4x96)
Download datasheet
130-094-256
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| ProCatch Glutathione Resin |
- 10 mL
Download datasheet
130-092-187
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- 25 mL
Download datasheet
130-092-186
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- 100 mL
Download datasheet
130-092-185
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| References |
| 1. Nakamura et al. (2010) Biol. Reprod. 82: 504-515. |
| 2. Paz et al. (2005) Mol Cancer Ther 4(11): 1801-1809. |
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