GST pulldown assays were performed as previously described (Shestakova et al., 2010 (link); Adell et al., 2014 (link)). Pierce magnetic glutathione beads (Thermo, 78602) were incubated in 0.1% BSA in ATPase buffer overnight. 5μg of GST-tagged proteins were bound to beads for 2 hours at 4°C, washed and optionally incubated with 500 ng of Cmp7-3xFLAG and/or Vps32-3xMyc for 1 hour at 4°C, followed by 5 five washing steps in ATPase buffer (+/- ATP) with 600mM NaCl. 500 ng of Vps4E233Q - 3xHA or Vps4-3xHA was added in the presence or absence of 1mM ATP for 10 minutes at room temperature. After three washing steps in ATPase buffer with 300mM NaCl, proteins were eluted from beads using sample buffer (2% SDS, 100mM Tris 6.8, 10% glycerol, 5% beta-mercaptoethanol, 0.01% bromophenol blue) at 96°C for 10 minutes. Samples were separated on a 12.5% SDS Page. Proteins were either stained by Brilliant Blue Coomassie or subjected to Western blotting.
Pierce glutathione magnetic beads
Manufactured by Thermo Fisher Scientific
Pierce Glutathione Magnetic Beads are a versatile tool for affinity purification of recombinant proteins containing a glutathione S-transferase (GST) tag. The beads are made of superparamagnetic iron oxide particles coated with glutathione, allowing for efficient capture and recovery of GST-tagged proteins from cell lysates or other samples.
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9 protocols using pierce glutathione magnetic beads
1
GST Pulldown Assay for Protein Interactions
2
GST Pulldown Assay for Protein Interactions
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GST pulldown assays were performed as previously described (Shestakova et al., 2010 (link), Adell et al., 2014 (link)). Pierce magnetic glutathione beads (Thermo, 78602) were incubated in 0.1% BSA in ATPase buffer overnight. 5μg of GST-tagged proteins were bound to beads for 2 hours at 4°C, washed and optionally incubated with 500 ng of Cmp7-3xFLAG and/or Vps32-3xMyc for 1 hour at 4°C, followed by 5 five washing steps in ATPase buffer (+/- ATP) with 600mM NaCl. 500 ng of Vps4E233Q -3xHA or Vps4-3xHA was added in the presence or absence of 1mM ATP for 10 minutes at room temperature. After three washing steps in ATPase buffer with 300mM NaCl, proteins were eluted from beads using sample buffer (2% SDS, 100mM Tris 6.8, 10% glycerol, 5% beta-mercaptoethanol, 0.01% bromophenol blue) at 96°C for 10 minutes. Samples were separated on a 12.5% SDS Page. Proteins were either stained by Brilliant Blue Coomassie or subjected to Western blotting.
3
Depletion of ATM and ATR Proteins
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For ATM and ATR depletion, pierce glutathione magnetic beads (Thermo Fisher Scientific) were conjugated with GST-Mre11 or ATRIP protein according to the manufacturer’s protocol. Beads were then added to extracts and removed with a magnet after incubation for 30 min. The remaining extract after beads removal was used as for the repair assay, The efficiency of ATM or ATR depletion was assessed by immunoblotting. Mock-depleted extract was prepared similarly with pierce glutathione magnetic beads that were not conjugated with Mre11 or ATRIP protein.
4
Enrichment of Ubiquitinated Proteins
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Kelly cells were treated with either DMSO or 1 μM CYC065 for the indicated times, lysed in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol and 200 μg/mL GST-TUBE2 (UM102; Biosensors, 2BScientific; TUBE indicates tandem ubiquitin binding entity) or in the absence of GST-TUBE2 for control pulldown. Pierce Glutathione Magnetic Beads (88821; Thermo Scientific Fisher) were used to pull down ubiquitinated proteins from cell lysates according to the manufacturer’s instructions. Ubiquitinated proteins were eluted by boiling beads Laemmli buffer and resolved by SDS-PAGE.
5
Aptamer Selection for Human DNMT1
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For the SELEX strategy, GST-tagged DNMT1 was used as target for the selection. Recombinant Human DNMT1 with an N-terminal GST tag was purchased from Active Motif and Pierce Glutathione Magnetic Beads (Thermo Scientific) were used to separate aptamer-GST-tagged protein complexes.
Before each cycle of SELEX, the 2′F-Py RNA pool was dissolved in RNAse free water and subjected to denaturation/renaturation steps of 85 °C for 5 min, ice for 2 min and 37 °C for 5 min. The RNA-protein incubation was performed in Binding Buffer (BB: 5 mM Tris-HCl pH 7,5; 5 mM MgCl2; 1 mM DTT; 100 mM NaCl). At each cycle, the pool was first incubated for 30 min with Glutathione Magnetic Beads with a gentle rotation, as counter-selection step, and then the unbound RNA was recovered on a magnetic separator and used for selection. The recovered sequences were incubated with GST-tagged DNMT1 at room temperature for 30 min with a gentle rotation. Aptamer-protein complexes were purified on magnetic beads. The unbound was removed on a magnetic separator and the beads containing RNA-protein complexes were washed with BB. Bound RNAs were recovered by TriFast (Euroclone) extraction and RT-PCR and finally transcribed for the following round.
Before each cycle of SELEX, the 2′F-Py RNA pool was dissolved in RNAse free water and subjected to denaturation/renaturation steps of 85 °C for 5 min, ice for 2 min and 37 °C for 5 min. The RNA-protein incubation was performed in Binding Buffer (BB: 5 mM Tris-HCl pH 7,5; 5 mM MgCl2; 1 mM DTT; 100 mM NaCl). At each cycle, the pool was first incubated for 30 min with Glutathione Magnetic Beads with a gentle rotation, as counter-selection step, and then the unbound RNA was recovered on a magnetic separator and used for selection. The recovered sequences were incubated with GST-tagged DNMT1 at room temperature for 30 min with a gentle rotation. Aptamer-protein complexes were purified on magnetic beads. The unbound was removed on a magnetic separator and the beads containing RNA-protein complexes were washed with BB. Bound RNAs were recovered by TriFast (Euroclone) extraction and RT-PCR and finally transcribed for the following round.
6
Expression and Purification of Human Ago2
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The cDNA for MID-domain of human Ago2 (His-Sumo-tagged) was provided by Bhushan Nagar (McGill University). Human eIF1A and mutants of eIF1A-ND/-CD/-GD were inserted into EcoRI/NotI sites of pGEX-4E-1 vectors with GST fusion at the N-term. Human Ago2 (full length) and fragments of L1(172 ~ 227aa), PAZ(227 ~ 349aa), MID (432 ~ 575aa) and PIWI (590 ~ 816aa) were inserted into NdeI/BamHI sites of a pET-6H2 vector. His-tagged proteins expressed in BL21(DE3) cells were extracted by sonication with lysis buffer (50mM Tris, 350mM NaCl, 10mM Imidazole, pH8.0, 20μM Benzonase nuclease, 10μg/ml RNase A, 100μg/ml lysozyme, 2% NP-40, 0.05% β-mercaptoethanol and protease inhibitor) followed by Ni-agarose resin (Qiagen) purification. GST-tagged proteins expressed in BL21(DE3) cells were extracted by MagneGST™ Protein Purification System (Promega) or Pierce Glutathione Magnetic Beads (Thermo Scientific).
7
Glutathione Bead Purification Protocol
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One hundred microliter translation mixture was diluted six times with Binding/Wash Buffer (125 mM Tris-HCl pH 8, 150 mM NaCl, 0.5% Triton-X), mixed with 12.5 μl equilibrated Pierce Glutathione Magnetic Beads (Thermo Scientific) and incubated on a rotator for 1 h at room temperature. The beads were washed three times with Binding/Wash buffer and resuspended in 50 μl PBS.
8
Expression and Purification of Human Ago2
9
GST-Ras Pull-Down Assay in HEK293 Cells
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HEK 293 BRI3BP-HA cells were transfected with a pEBG vector expressing GST control or GST-Ras fusion proteins, wild type or mutant. In pEBG, a GST fusion protein is expressed in mammalian cells under control of an EF1alpha promoter (49) (link).
GST is fused to the N-terminus of the Ras protein. Per pull-down, 3 wells of a 12-well dish were each transfected with 1 µg DNA. 24 hours after transfection, extracts were prepared in Tris Lysis Buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 1% Triton, 5% glycerol) plus 1 µg/ml Leupeptin, 1 mM PMSF, and 1 mM DTT. GST proteins were captured on Pierce glutathione magnetic beads (Thermo Fisher Scientific) for 1 hour, then washed twice in Tris Lysis Buffer, twice in Tris Buffered Saline with 287 mM NaCl and 0.1% Tween 20 (TBST), and twice in TBST with 137 mM NaCl. Each experiment was repeated 3 times, except for K-Ras4A-BRI3BP interaction, which was n=2.
GST is fused to the N-terminus of the Ras protein. Per pull-down, 3 wells of a 12-well dish were each transfected with 1 µg DNA. 24 hours after transfection, extracts were prepared in Tris Lysis Buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 1% Triton, 5% glycerol) plus 1 µg/ml Leupeptin, 1 mM PMSF, and 1 mM DTT. GST proteins were captured on Pierce glutathione magnetic beads (Thermo Fisher Scientific) for 1 hour, then washed twice in Tris Lysis Buffer, twice in Tris Buffered Saline with 287 mM NaCl and 0.1% Tween 20 (TBST), and twice in TBST with 137 mM NaCl. Each experiment was repeated 3 times, except for K-Ras4A-BRI3BP interaction, which was n=2.
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