Protein G dynabeads™ (ThermoFisher, 10004D) were mixed with the corresponding antibody overnight at 4°C. For Co-IP assays, cells were incubated in NP40 lysis buffer (Beyotime, P0013F) containing PMSF (Beyotime, ST506-2). The following day cell lysates containing the antigen was added to the Protein G mixture. The bead-Ab-Ag complexes were washed five times by gentle pipetting and subjected to immunoblotting. For the pull-down assays, His-labeled VP1, VP2, and VP3, pGEX-6P-1, and GST-TMEM39A were expressed in E.coli BL21 strain. GST alone or GST-TMEM39A protein was mixed with the glutathione agarose resin (Thermo Fisher, 21516) and incubated overnight at 4°C. The following day, His-labeled proteins were added separately to the above mixture. After conjugation, the unbound proteins were washed away and subjected to immunoblotting.
Glutathione agarose resin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Glutathione agarose resin is a chromatography medium used for the purification of proteins that contain a glutathione-S-transferase (GST) tag. It consists of glutathione, a tripeptide, covalently coupled to agarose beads. This resin can be used to selectively bind and capture GST-tagged proteins from complex mixtures, allowing for their subsequent isolation and purification.
Automatically generated - may contain errors
Lab products found in correlation
Amylose resin, by New England Biolabs (3 mentions)
Np 40 lysis buffer, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
E coli bl21 de3 cells, by New England Biolabs (1 mentions)
C3 emulsiflex, by Roche (1 mentions)
Superdex 75, by GE Healthcare (1 mentions)
Source s, by GE Healthcare (1 mentions)
Pgex 6p 1 vector, by GE Healthcare (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Roche (1 mentions)
Ni nta agarose resin, by Thermo Fisher Scientific (1 mentions)
Ktapurifier upc 10, by GE Healthcare (1 mentions)
E coli bl21 de3, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Hispur cobalt resin, by Thermo Fisher Scientific (1 mentions)
Q tof premier, by Waters Corporation (1 mentions)
Superdex 75 10 300, by GE Healthcare (1 mentions)
Ni nta bead, by Qiagen (1 mentions)
Hrv 3c protease, by Thermo Fisher Scientific (1 mentions)
16 protocols using glutathione agarose resin
1
Co-immunoprecipitation and Pull-down Assays
2
Purification of BRG1 AT-BD and BD
3
Purification of His-tagged and GST-tagged Proteins
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N-terminally hexahistidine-tagged proteins expressed in pQE30 or pET21a were purified from Escherichia coli TG1 or BL21/DE3/pLysS cells. Single colonies were picked into Luria-Bertani broth and grown at 37°C to an OD600 of 0.6. Protein expression was induced by addition of isopropyl β-D-1-thiogalactopyranoside (to 0.1 mM) and continued for 2 h at 37°C (or overnight at 22°C) after which cells were pelleted and resuspended in lysis buffer (20 mM Tris, pH 7.5, 20 mM imidazole, 0.5 mM MgCl2, 1.4 mM 2-mercaptoethanol, 0.05% Tween 20, 500 mM NaCl, 0.1 mg/ml lysozyme). Cells were incubated on ice for 30 min and sonicated to complete lysis. Protein purification was performed using Ni-nitrilotriacetic acid (Ni-NTA; Qiagen) according to standard procedures (29 (link)). N-terminally glutathione-S-transferase (GST)-tagged nsp1β was expressed in BL21/DE3/pLysS cells as above and purified using glutathione agarose resin (ThermoFisher Scientific) according to standard procedures (30 (link)). Proteins were dialysed against 50 mM Tris, pH 7.5, 100 mM KCl, 1 mM dithiothreitol (DTT), 0.05 mM ethylenediaminetetraacetic acid (EDTA) and 5% glycerol, quantified by bicinchoninic acid assay (Pierce) and stored at −80°C until required.
4
Purification of LOTUS and Vasa Proteins
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cDNA fragments of LOTUS domains were obtained by PCR amplification or gene synthesis (GenScript) and subcloned into pET28a (His-tag) vector (Supplementary Table S2 ). Drosophila Vasa (200–661aa) cDNA was obtained by PCR amplification and were subcloned into pGEX-4t-1 (GST-tag) vector. Plasmids were transformed into Escherichia coli (BL21 or Rosetta) and recombinant proteins were induced with 0.2 mM IPTG (Roche) at 18°C overnight. His-tagged proteins were affinity purified with Ni-NTA Agarose Resin (Thermo Scientific). GST-tagged proteins were affinity purified using Glutathione Agarose Resin (Thermo Scientific). Proteins were further purified by gel filtration chromatography with ÄKTApurifier UPC 10 (GE Healthcare). The running buffer for gel filtration chromatography is 10 mM Tris–HCl (pH 7.4) and 100 mM KCl.
5
GST-tagged Protein Pulldown Assay with RAD51
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30 μg of BRC3-GST and Ex27-GST was incubated with 200 μl of glutathione agarose resin (Thermo Fisher) in T100 buffer (25 mM Tris-HCl, pH = 7.5, 100 mM KCl, 10 % glycerol, 0.5 % EDTA, 1 mM DTT, 0.01 % NP40) for 2 hr at 4°C with shaking. Beads were then washed twice with 1 mL of T100 buffer to eliminate unbound protein. Subsequently, 10 μL of beads coated with Ex27-GST or BRC3-GST were incubated with 2 μM RAD51 or its variants in buffer T100 in 20 μl final volume for 1 hr at 4°C. Following the incubation, supernatant was removed and beads were washed twice with 500 μL of T100 buffer. Beads were boiled in Laemmli buffer to obtain the bound fraction. Samples were loaded onto 12 % SDS-PAGE gel and stained with Coomassie Blue.
6
Affinity Purification of NS4B-Flag
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Plasmids pGEX-6p-1 and pGEX-GST-Rab5 were transformed into E. coli BL21 (DE3) (Invitrogen, Carlsbad, CA, United States) to express GST and GST-Rab5 proteins, respectively. Glutathione agarose resin (Thermo Scientific, #21516) balanced with equilibrium liquid (TBS and Pull-Down lysis buffer at a ratio of 1:1) was utilized to combine the proteins into complexes according to the manufacturers’ direction. Then, the complexes were incubated with NS4B-Flag expressed by HEK-293T cells transfected with plasmid pcDNA-NS4B-Flag at 4°C for 2 h. After that, elution buffer (1 mL TBS containing 3.1 g glutathione) was used to separate the combined proteins and the eluted proteins were analyzed by western blot.
7
Purification and Deimination Assay of GST-PADI4
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Purification of recombinant GST-PADI4 and in vitro deimination assays were carried out essentially as described (Cuthbert et al. 2004 (link)). Briefly, GST-PADI4 was overexpressed from a pGEX6P construct at 30°C in 2TY broth by the addition of 0.1 mM IPTG and purified using glutathione agarose resin (Thermo Scientific). Deimination reactions were carried out using bead-bound PADI4 in 50 mM Tris, pH 7.5, 2 mM DTT and complete protease inhibitor cocktail (Roche), in the presence or absence of 2 mM CaCl2 at 30°C for 1 h.
8
Recombinant Protein Expression and Purification
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The full-length coding sequences of OsEDS1 and OsCATC were inserted into the pMAL or pGEXT-6p-1 vectors, respectively, and transformed into Escherichia coli strain BL21 to express MBP-OsEDS1 and GST-OsCATC. These recombinant proteins were induced by 1 mM isopropyl β-D-1-thiogalactopyranoside at 16 °C for 20 h. MBP-OsEDS1 was purified with Amylose Resin (New England Biolabs, USA) and GST-OsCATC was purified with Glutathione Agarose Resin (Thermo Scientific, USA) according to the manufacturer's instructions. Primers used for plasmid construction are listed in Supplemental Table S1 .
9
Affinity Purification of Tagged Proteins
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HIS-tagged, GST-tagged, and MBP-tagged proteins were expressed in BL21(DE3) Escherichia coli transformed with the corresponding plasmids by overnight induction at 23°C with 1 mM isopropyl-β-d-1-thio-galactopyranoside (IPTG). For MBP-tagged proteins, bacterial growth media was supplemented with 0.2% (weight/vol) glucose. Bacteria pelleted from 1 liter of culture were resuspended in 25 ml of buffer (50 mM NaH2PO4, pH 7.4, 300 mM NaCl, 10 mM imidazole, 1% [vol/vol] Triton X-100 supplemented with protease inhibitor cocktail [leupeptin 1 μM, pepstatin 2.5 μM, aprotinin 0.2 μM, phenylmethylsulfonyl fluoride 1 mM]). After four pulses of sonication (20 second each with 1 minute rest on ice), lysates were centrifuged at 12,000 × g for 20 min at 4°C. The soluble fraction of the lysate was used for affinity purification on HisPur Cobalt resin (Thermo, Cat#89964), Glutathione Agarose resin (Thermo, Cat#16100) or Amylose Resin (New England Biolabs, E8021), depending of the fusion tag, and eluted with 50 mM Tris-HCl (pH 8), 100 mM NaCl supplemented with either 250 mM imidazole, or 30 mM reduced glutathione, or 20 mM amylose, respectively. Proteins were dialyzed overnight at 4°C against PBS. All protein samples were aliquoted and stored at −80°C.
10
Isotopic Labeling of Histone H4
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The histone H4 tail (residues 1–25 followed by a C-terminal tyrosine for quantification) was expressed from pGEX6p as a fusion with an N-terminal GST tag followed by a PreScission protease cleavage site (Supplementary file 2A ). This was overexpressed in E. coli BL21 (DE3) (NEB) grown in M9 minimal media supplemented with vitamin (Centrum daily multivitamin), 1 g/L 15NH4Cl, and 5 g/L D-glucose. Cells were grown to OD600 ~1.0 and induced with 0.5 mM IPTG at 37 °C for 4 hr. The 15N-GST-H4 peptide fusion was purified on glutathione agarose resin (Thermofisher Scientific), cleaved with PreScission protease (16 hr at 4 °C), and products resolved by SEC (Superdex 75 10/300; GE Healthcare Life Sciences). Peptide identity was validated by mass spectrometry with positive electrospray ionization (Waters Q-Tof Premier). Samples were diluted 1:2 or 1:4 in water/acetonitrile (1:1) with 0.1% formic acid. Acquisition and deconvolution software used during data collection and analysis were MassLynx and MaxEnt, respectively. 15N-H4 (1-25) peptide concentration was determined by UV-vis spectroscopy using the non-native C-terminal tyrosine.
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