Gstrap ff affinity columns

Manufactured by GE Healthcare

Sourced in United States

GSTrap FF affinity columns are designed for the purification of recombinant proteins containing a glutathione S-transferase (GST) tag. The columns utilize immobilized glutathione to capture GST-tagged proteins, which can then be eluted under mild, non-denaturing conditions.

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Lab products found in correlation

γ 32p atp, by PerkinElmer (4 mentions) Kinase buffer, by New England Biolabs (2 mentions) Recombinant cdk1 cyclin b complex, by New England Biolabs (2 mentions) Cdk1 cyclin b, by Sino Biological (2 mentions) Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions) L arabinose, by Merck Group (1 mentions)

Close spelling variants of this product

GSTrap FF column GSTrap affinity column GSTrap FF GSTrap column

5 protocols using gstrap ff affinity columns

In Vitro Phosphorylation of MST2

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GST-tagged MST2 or MST2-S385A (cloned in pGEX-5X-1) was bacterially expressed and purified on GSTrap FF affinity columns (GE Healthcare) following the manufacturer’s instructions. GST-MST2 (1 μg) was incubated with 5-10 U recombinant CDK1/cyclin B complex (New England Biolabs) or 50-100 ng CDK1/cyclin B (SignalChem) in kinase buffer (New England Biolabs) in the presence of 5 μCi γ-³²P-ATP (3000 Ci/mmol, PerkinElmer) as we previously described [15 (link)]. Active CDK2, CDK5, p38, JNK1, JNK2, MEK1, ERK1, and PLK1 kinases were also purchased from SignalChem.

Chen X., Chen Y, & Dong J. (2016). MST2 Phosphorylation at Serine 385 in Mitosis Inhibits its Tumor Suppressing Activity. Cellular signalling, 28(12), 1826-1832.

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Purification of GST and His-tagged PACS-2 FBR

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Plasmids expressing GST, GST-PACS-2FBR_38–202 corresponding to residues 38–202 (cargo/adaptor-binding region-FBR) [31] (link) and pET15b-FBR2 plasmid expressing His₆-PACS-2FBR_38–217[22] (link) were induced in BL21-A1 E. coli (Invitrogen) with 1 mM isopropyl-β-D-thiogalactoside (Calbiochem, Gibbstown, NJ) and 0.2% L-arabinose (Sigma Aldrich) for 4 hours at 37°C. Bacterial pellets were resuspended in PBS (pH 7.4) containing 1 mM PMSF and 1 mM DTT, subjected to sonication, and incubated on ice for 30 min in the presence of 1% Triton X-100. Soluble material was recovered by centrifugation at 13,000×g for 20 min at 4°C and subsequently purified using GSTrap FF affinity columns (GE Healthcare, Piscataway, NJ) or HisPur Ni-NTA Spin Columns (Thermo Scientific/Pierce Biotechnology, Rockford, IL) following the manufacturer's instructions.

Guicciardi M.E., Werneburg N.W., Bronk S.F., Franke A., Yagita H., Thomas G, & Gores G.J. (2014). Cellular Inhibitor of Apoptosis (cIAP)-Mediated Ubiquitination of Phosphofurin Acidic Cluster Sorting Protein 2 (PACS-2) Negatively Regulates Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Cytotoxicity. PLoS ONE, 9(3), e92124.

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Purification and Phosphorylation of YES Kinase

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YES cDNA (encodes amino acids 1–300 of YES) was cloned into the pGEX-5X-1 vector. The glutathione S-transferase (GST)-tagged proteins were bacterially expressed and purified on GSTrap FF affinity columns (GE Healthcare, IL, USA) following the manufacturer’s instructions. GST-YES or GST-YES-5A (0.5–1 µg each) was incubated with 10 U recombinant CDK1/cyclin B complex (New England Biolabs, MA, USA) in kinase buffer [15 (link)] in the presence of 5 µCi γ−³²P-ATP (3000 Ci/mmol, PerkinElmer, MA, USA). The samples were resolved by SDS-PAGE, transferred onto PVDF (Millipore, MA, USA) and visualized by autoradiography followed by Western blotting or detected by phospho-specific antibodies.

Wang Z., Chen X., Zhong M.Z., Yang S., Zhou J., Klinkebiel D.L., Karpf A.R., Chen Y, & Dong J. (2018). Cyclin-Dependent Kinase 1-Mediated Phosphorylation of YES Links Mitotic Arrest and Apoptosis during Antitubulin Chemotherapy. Cellular signalling, 52, 137-146.

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Phosphorylation Assay for GST-PBK

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GST-tagged PBK and PBK-4A were cloned in pGEX-5X-1 and proteins were bacterially expressed and purified on GSTrap FF affinity columns (GE Healthcare) following the manufacturer’s instructions. GST-PBK (0.5–1 μg) was incubated with 10 U recombinant CDK1/cyclin B complex (New England Biolabs) or 100 ng CDK1/cyclin B (SignalChem) in kinase buffer (New England Biolabs) in the presence of 5 μCi γ-³²P-ATP (3000 Ci/mmol, PerkinElmer). Phosphorylation (³²P incorporation) was visualized by autoradiography followed by Western blotting or detected by phospho-specific antibodies.

Stauffer S., Zeng Y., Zhou J., Chen X., Chen Y, & Dong J. (2017). CDK1-mediated Mitotic Phosphorylation of PBK is Involved in Cytokinesis and Inhibits its Oncogenic Activity. Cellular signalling, 39, 74-83.

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In vitro Phosphorylation of GST-SET Proteins

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The glutathione S-transferase (GST)-tagged proteins were bacterially expressed and purified on GSTrap FF affinity columns (GE Healthcare, Chicago, IL, USA) following the manufacturer’s instructions. About 0.5 µg of GST-SET proteins were incubated with 10 U recombinant CDK1/cyclin B1 complex (New England Biolabs, Ipswich, MA, USA) in the presence of 5 µCi γ-³²P-ATP (3000 Ci/mmol, PerkinElmer, Waltham, MA, USA). Purified CDK1/cyclin B1 complex from SignalChem (Richmond, BC, Canada) was also used for in vitro kinase assays using phospho-specific antibodies. The samples were resolved by SDS-PAGE, transferred onto PVDF (Millipore, Burlington, MA, USA), and visualized by autoradiography or detected by phospho-specific antibodies.

Yin L., Zeng Y., Xiao Y., Chen Y., Shen H, & Dong J. (2019). Cyclin-dependent kinase 1-mediated phosphorylation of SET at serine 7 is essential for its oncogenic activity. Cell Death & Disease, 10(6), 385.

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