Glutathione Agarose


Agarose Guide
The GST tag is a 26 kDa protein that binds to glutathione-coupled agarose resins. This tag is considerably larger than the His tag and is often chosen to promote recombinant protein folding. PureCube Glutathione Agarose is based on BioWorks Workbeads. For purification from cell culture supernatants or pull-down experiments, we recommend PureCube Glutathione MagBeads. To detect GST-tagged proteins in Western Blot experiments, Cube Biotech offers a highly specific GST antibody.

PureCube Glutathione Agarose provides:


GST / Gluthathione-affinity agarose resins from Cube Biotech were successfully used in the following publications:

 ProteinYearAuthor
Human LGP2, RIG‐I, MDA5 and LGP2 CTD 2018   van der Veen A.G., Maillard P.V., Schmidt J.M., Lee S.A., Deddouche-Grass S., Borg A., Kjaer S., Snijders A.P., Reis e Sousa C.1
Human DNA polymerase α-primase and B- fusion proteins 2017  Kilkenny M.L., Simon A.C., Mainwaring J., Wirthensohn D., Holzer S., Pellegrini L.2
ISG15 2018  Napolitano A., van der Veen A.G., Bunyan M., Borg A., Frith D., Howell S., kjaer S., Beling A., Snijders A.P., Knobeloch K-P., Frickel E-M.3
Ran (wt) Ran KxQ/R; NTF2; RCC1 49-444; Importin- beta; Ran KxAcK 2019  de Boor S.4
several TBX3 fusion proteins 2015  Fischer K.5
GtPEBB, mGtPEBB and variants 2019 Sommerkamp J.A.; Frankenberg-Dinkel N.; Hofmann E.6

PureCube GST Affinity resins compared to other suppliers

High protein binding capacity
In a comparative study with equivalent, market-leading resins, PureCube Glutathione Agarose proved to be a competitive alternative, exhibiting the same protein binding capacity. Figure 1 is an SDS-PAGE of GST purified on gravity columns packed with PureCube Glutathione Agarose and the equivalent resin from Competitor G. Both resins yielded up to 10 mg/mL protein. TL: total lysate; CL: cleared lysate; FT: flow-through; W1/W4: wash fractions; E1-E6: elution fractions.
Glutathione Binding affinity
Fig. 1: Purification yield is comparable to that of a market-leading resin. SDS-PAGE of GST expressed in E.coli and purified in gravity columns with PureCube Glutathione Agarose and the equivalent resin from Competitor G. Up to 10 mg/ml protein yield was obtained with PureCube Glutathione Agarose (elution fractions E1-E6, Cube)

Features

Usage Specific binding and purification of GST-tagged proteins
Specifity Affinity to GST-tagged proteins
High binding capacity 10 mg / ml
Filling quantity Delivered as a 50 % suspension
Bead size 40 µm
Bead Ligand Affinity agarose with GST-Antibody
Required equipment
 
  • Lysis Buffer
  • Wash Buffer
  • Elution Buffer
  • Ice bath
  • Refrigerated centrifuge for 50 mL tube (min 10,000 x g)
  • 50 mL centrifuge tube
  • Micropipettor and Micropipetting tips
  • Disposable gravity flow columns with capped bottom outlet, 2 ml
  •  pH meter
  • Optional: 15 mL conical propylene tubes pH meter
  •  UV/VIS Spectrophotometer
  • End-over-end shaker
  • SDS-PAGE buffers, reagents and equipment
  •  Optional: Western Blot reagents and equipment for control purposes using GST-Antibody.

Applications

All protocols and buffer compositions are also avaible as PDF-Files on the Protocols & Datasheets page.
   
A.Protein purification protocol
 
 
  1. Thaw the E. coli cell pellets corresponding to 200 mL bacterial culture on ice for 15 min. Optional: Freezing the cell pellet at -20 °C for 30 min prior to incubation at room temperature improves lysis by lysozyme.
  2. Resuspend the cell pellet in 10 mL Lysis Buffer and pour it into a 50 mL conical centrifuge tube. If the solution is very viscous, add 3 units Benzonase® per mL E.coli culture volume to the lysis buffer. Alternatively or additionally, sonicate the lysate to improve cell disruption.
  3. Incubate on an end-over-end shaker at room temperature for 30 min, or at 4 °C for 1 h, depending on the temperature stability of the protein.
  4. Centrifuge the lysate at 10.000 x g for 30 min at 2-8 °C and carefully collect the supernatant without touching the pellet. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  5. Resuspend the PureCube Glutathione Agarose by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50 % suspension (corresponding to 500 μL bed volume) into a 15 mL conical centrifuge tube. Allow the resin to settle by gravity and remove the supernatant. Tip: Alternatively, resin equilibration can be performed directly in the disposable gravity flow column.
  6. Add 5 mL of Wash Buffer and gently resupend the suspension to equilibrate the resin. Allow the resin to settle by gravity and remove the supernatant.
  7. Add the cleared lysate prepared in step 4 and incubate at 4 °C for 1 h on an end-over-end shaker. Tip: Alternatively, batch binding can be performed directly in a gravity flow column with closed bottom and top outlets.
  8. Pour the complete suspension into a disposable gravity flow column with a capped bottom outlet.
  9. Remove the bottom cap of the column and collect the flow-through.
  10. Wash twice with 2.5 mL each of Wash Buffer.
  11. Optional: To remove contaminants such as chaperones, perform an additional wash step with ATP buffer.
  12. Elute the GST-tagged protein by adding 0.5 mL Elution Buffer.
  13. Repeat step 11 five times, for a total of six elutions. Collect each elution fraction separately. Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields.
  14. Determine the protein concentration of the elution fractions with Bradford assay, using BSA as protein standard.
  15. 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.
  16. Optional: Perform Western Blot experiment using GST Antibody.

References:

1. van der Veen, A.G. et al. “The RIG-I-like receptor LGP2 inhibits Dicer-dependent processing of long double-stranded RNA and blocks RNA interference in mammalian cells.” The EMBO journal vol. 37,4 (2018): e97479. doi:10.15252/embj.201797479
2. Kilkenny, M. L et al. “The human CTF4-orthologue AND-1 interacts with DNA polymerase α/primase via its unique C-terminal HMG box.” Open biology vol. 7,11 (2017): 170217. doi:10.1098/rsob.170217.
3. Napolitano, Anna et al. “Cysteine-Reactive Free ISG15 Generates IL-1β-Producing CD8α+ Dendritic Cells at the Site of Infection.” Journal of immunology (Baltimore, Md. : 1950) vol. 201,2 (2018): 604-614. doi:10.4049/jimmunol.1701322
4. Boor, Susanne de (2015). Mechanistic studies on the regulation of Ran-function by post-translational lysine-acetylation. Dissertation, Universität zu Köln.
5. Fischer K. (2015). TBX3-Mutationsanalysen und Konstruktion einer Zelllinie für TBX2-Inhibitorscreenings. Dissertation, Universität zu Mainz.
6. A. Sommerkamp, et al. (2019). Crystal structure of the first eukaryotic bilin reductase Gt PEBB reveals a flipped binding mode of dihydrobiliverdin. Journal of Biological Chemistry. jbc.RA119.009306. 10.1074/jbc.RA119.009306.
Agarose Guide
The GST tag is a 26 kDa protein that binds to glutathione-coupled agarose resins. This tag is considerably larger than the His tag and is often chosen to promote recombinant protein folding. PureCube Glutathione Agarose is based on BioWorks Workbeads. For purification from cell culture supernatants or pull-down experiments, we recommend PureCube Glutathione MagBeads. To detect GST-tagged proteins in Western Blot experiments, Cube Biotech offers a highly specific GST antibody.

PureCube Glutathione Agarose provides:


GST / Gluthathione-affinity agarose resins from Cube Biotech were successfully used in the following publications:

 ProteinYearAuthor
Human LGP2, RIG‐I, MDA5 and LGP2 CTD 2018   van der Veen A.G., Maillard P.V., Schmidt J.M., Lee S.A., Deddouche-Grass S., Borg A., Kjaer S., Snijders A.P., Reis e Sousa C.1
Human DNA polymerase α-primase and B- fusion proteins 2017  Kilkenny M.L., Simon A.C., Mainwaring J., Wirthensohn D., Holzer S., Pellegrini L.2
ISG15 2018  Napolitano A., van der Veen A.G., Bunyan M., Borg A., Frith D., Howell S., kjaer S., Beling A., Snijders A.P., Knobeloch K-P., Frickel E-M.3
Ran (wt) Ran KxQ/R; NTF2; RCC1 49-444; Importin- beta; Ran KxAcK 2019  de Boor S.4
several TBX3 fusion proteins 2015  Fischer K.5
GtPEBB, mGtPEBB and variants 2019 Sommerkamp J.A.; Frankenberg-Dinkel N.; Hofmann E.6

PureCube GST Affinity resins compared to other suppliers

High protein binding capacity
In a comparative study with equivalent, market-leading resins, PureCube Glutathione Agarose proved to be a competitive alternative, exhibiting the same protein binding capacity. Figure 1 is an SDS-PAGE of GST purified on gravity columns packed with PureCube Glutathione Agarose and the equivalent resin from Competitor G. Both resins yielded up to 10 mg/mL protein. TL: total lysate; CL: cleared lysate; FT: flow-through; W1/W4: wash fractions; E1-E6: elution fractions.
Glutathione Binding affinity
Fig. 1: Purification yield is comparable to that of a market-leading resin. SDS-PAGE of GST expressed in E.coli and purified in gravity columns with PureCube Glutathione Agarose and the equivalent resin from Competitor G. Up to 10 mg/ml protein yield was obtained with PureCube Glutathione Agarose (elution fractions E1-E6, Cube)

Features

Usage Specific binding and purification of GST-tagged proteins
Specifity Affinity to GST-tagged proteins
High binding capacity 10 mg / ml
Filling quantity Delivered as a 50 % suspension
Bead size 40 µm
Bead Ligand Affinity agarose with GST-Antibody
Required equipment
 
  • Lysis Buffer
  • Wash Buffer
  • Elution Buffer
  • Ice bath
  • Refrigerated centrifuge for 50 mL tube (min 10,000 x g)
  • 50 mL centrifuge tube
  • Micropipettor and Micropipetting tips
  • Disposable gravity flow columns with capped bottom outlet, 2 ml
  •  pH meter
  • Optional: 15 mL conical propylene tubes pH meter
  •  UV/VIS Spectrophotometer
  • End-over-end shaker
  • SDS-PAGE buffers, reagents and equipment
  •  Optional: Western Blot reagents and equipment for control purposes using GST-Antibody.

Applications

All protocols and buffer compositions are also avaible as PDF-Files on the Protocols & Datasheets page.
   
A.Protein purification protocol
 
 
  1. Thaw the E. coli cell pellets corresponding to 200 mL bacterial culture on ice for 15 min. Optional: Freezing the cell pellet at -20 °C for 30 min prior to incubation at room temperature improves lysis by lysozyme.
  2. Resuspend the cell pellet in 10 mL Lysis Buffer and pour it into a 50 mL conical centrifuge tube. If the solution is very viscous, add 3 units Benzonase® per mL E.coli culture volume to the lysis buffer. Alternatively or additionally, sonicate the lysate to improve cell disruption.
  3. Incubate on an end-over-end shaker at room temperature for 30 min, or at 4 °C for 1 h, depending on the temperature stability of the protein.
  4. Centrifuge the lysate at 10.000 x g for 30 min at 2-8 °C and carefully collect the supernatant without touching the pellet. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  5. Resuspend the PureCube Glutathione Agarose by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50 % suspension (corresponding to 500 μL bed volume) into a 15 mL conical centrifuge tube. Allow the resin to settle by gravity and remove the supernatant. Tip: Alternatively, resin equilibration can be performed directly in the disposable gravity flow column.
  6. Add 5 mL of Wash Buffer and gently resupend the suspension to equilibrate the resin. Allow the resin to settle by gravity and remove the supernatant.
  7. Add the cleared lysate prepared in step 4 and incubate at 4 °C for 1 h on an end-over-end shaker. Tip: Alternatively, batch binding can be performed directly in a gravity flow column with closed bottom and top outlets.
  8. Pour the complete suspension into a disposable gravity flow column with a capped bottom outlet.
  9. Remove the bottom cap of the column and collect the flow-through.
  10. Wash twice with 2.5 mL each of Wash Buffer.
  11. Optional: To remove contaminants such as chaperones, perform an additional wash step with ATP buffer.
  12. Elute the GST-tagged protein by adding 0.5 mL Elution Buffer.
  13. Repeat step 11 five times, for a total of six elutions. Collect each elution fraction separately. Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields.
  14. Determine the protein concentration of the elution fractions with Bradford assay, using BSA as protein standard.
  15. 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.
  16. Optional: Perform Western Blot experiment using GST Antibody.

References:

1. van der Veen, A.G. et al. “The RIG-I-like receptor LGP2 inhibits Dicer-dependent processing of long double-stranded RNA and blocks RNA interference in mammalian cells.” The EMBO journal vol. 37,4 (2018): e97479. doi:10.15252/embj.201797479
2. Kilkenny, M. L et al. “The human CTF4-orthologue AND-1 interacts with DNA polymerase α/primase via its unique C-terminal HMG box.” Open biology vol. 7,11 (2017): 170217. doi:10.1098/rsob.170217.
3. Napolitano, Anna et al. “Cysteine-Reactive Free ISG15 Generates IL-1β-Producing CD8α+ Dendritic Cells at the Site of Infection.” Journal of immunology (Baltimore, Md. : 1950) vol. 201,2 (2018): 604-614. doi:10.4049/jimmunol.1701322
4. Boor, Susanne de (2015). Mechanistic studies on the regulation of Ran-function by post-translational lysine-acetylation. Dissertation, Universität zu Köln.
5. Fischer K. (2015). TBX3-Mutationsanalysen und Konstruktion einer Zelllinie für TBX2-Inhibitorscreenings. Dissertation, Universität zu Mainz.
6. A. Sommerkamp, et al. (2019). Crystal structure of the first eukaryotic bilin reductase Gt PEBB reveals a flipped binding mode of dihydrobiliverdin. Journal of Biological Chemistry. jbc.RA119.009306. 10.1074/jbc.RA119.009306.
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PureCube Glutathione Agarose PureCube Glutathione Agarose
Article number: 32103
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Article number: 32103
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