To obtain GST-GATA3 fusion protein, human full-length GATA3 was subcloned into pGEX-4T-3 (Amersham, Piscataway, NJ, USA) and the construct was expressed in BL21DE3 E. coli. The expression of soluble GST-GATA3 was confirmed by mass spectrometry. E. coli lysates containing GST-GATA3 were incubated with Glutathione-Sepharose 4 Fast Flow beads (GE Healthcare Life Sciences) at 4 °C for 2 h to generate the GST-GATA3 conjugated beads. Lysates of cells expressing different constructs of HIF-1α were incubated with GST-GATA3 conjugated beads at 4 °C for 18 h and then washed with lysis buffer for five times. The pulled down proteins were eluted by SDS sample buffer and analysed by western blotting.
Glutathione sepharose 4 fast flow beads
Manufactured by GE Healthcare
Sourced in United States, Sweden
Glutathione Sepharose 4 Fast Flow beads are a chromatography medium designed for the purification of glutathione-binding proteins. The beads consist of crosslinked agarose with covalently coupled glutathione, which enables the specific capture and separation of proteins expressing glutathione-binding domains.
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Lab products found in correlation
Pharosfx imager, by Bio-Rad (6 mentions)
Complete mini edta free protease inhibitor cocktail, by Roche (6 mentions)
Tnt t7 coupled reticulocyte lysate system, by Promega (6 mentions)
Solubl21, by AMS Biotechnology (4 mentions)
Lds page sample buffer, by Thermo Fisher Scientific (2 mentions)
Tnt coupled reticulocyte lysate system, by Promega (2 mentions)
Anti ha agarose beads, by Merck Group (2 mentions)
Ha peptide, by Merck Group (2 mentions)
Bugbuster reagent, by Promega (2 mentions)
Fugene hd transfection reagent, by Promega (2 mentions)
Pgex 6p 1, by GE Healthcare (2 mentions)
Hitrap sp column, by GE Healthcare (2 mentions)
Pgex 4t 3, by Cytiva (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
E coli solubl21, by AMS Biotechnology (1 mentions)
His bind resin, by Merck Group (1 mentions)
D glucose, by Cambridge Isotopes (1 mentions)
38 protocols using glutathione sepharose 4 fast flow beads
1
Purification and Binding of GST-GATA3 Fusion
2
GST Pulldown Assay for Protein Interactions
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GST pulldown assays with in vitro–translated [35S]-labeled proteins were done as described previously (Kimura et al., 2015 (link); Chauhan et al., 2016 (link)). All GST-tagged recombinant proteins were expressed in Escherichia coli BL21(DE3) and/or SoluBL21 (Amsbio). GST fusion proteins were purified on Glutathione Sepharose 4 Fast-Flow beads (GE Healthcare). [35S]-labeled Myc-tagged proteins were cotranscribed/translated in vitro using the TnT T7–coupled reticulocyte lysate system (Promega). The in vitro–translated [35S]-labeled Myc-tagged proteins were then incubated with GST-tagged proteins in 250 µl of NETN-E buffer (50 mm Tris, pH 8.0, 100 mm NaCl, 6 mm EDTA, 6 mm EGTA, 0.5% NP-40, and 1 mm dithiothreitol supplemented with cOmplete mini EDTA-free protease inhibitor cocktail [Roche]) for 2 h at 4°C and then washed five times with 1 ml of NETN-E buffer, boiled with 2× SDS gel loading buffer, and subjected to SDS-PAGE. The separated proteins were then transferred to polyvinylidene difluoride membranes using the Trans-Blot Turbo Transfer system (Bio-Rad Laboratories). The GST-tagged proteins were detected by staining with Ponceau S, whereas the radiolabeled proteins were detected in a PharosFX imager (Bio-Rad Laboratories).
3
Bacterial Expression and Purification of GST-ECT
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To induce expression of GST-ECT protein, BL21 Escherichia coli cells transformed with pETM33-ECT were grown to an OD600 of 0.5 and treated with 100 μM (final concentration) Isopropyl β-D-1-thiogalactopyranoside (IPTG), prior to culture at 30 °C for 5 h. Cells were pelleted by centrifugation and pellets washed twice with ice-cold PBS. Bacterial pellets were resuspended in 25 ml of bacterial lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 2 mM EDTA (pH 8), 1% (v/v) Triton X-100, 1X complete protease inhibitor cocktail (Roche), 2 mM dithiothreitol, 0.25 mg/ml lysozyme (Sigma) and 10 μg/ml DNAseI (Sigma)] and subjected to at least three freeze-thaw cycles. Extracts were clarified by centrifugation and transferred to a fresh tube, with a small aliquot used to verify protein expression by Coomassie staining. In parallel, 1 ml of Glutathione Sepharose 4 Fast Flow beads (GE Healthcare) was washed in 5 ml bacterial lysis buffer. To generate GST-ECT beads, the beads were added to cleared bacterial lysate and incubated for 30 min at 4 °C on a rotator. Beads were washed five times in bacterial lysis buffer and resuspended in the same buffer supplemented with 50% glycerol, prior to storage at −20 °C. GST-ECT beads were equilibrated into brain lysis buffer before use in ECT pulldown experiments.
4
SNARE-Mediated Protein Interaction Assay
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GST-His6-tagged R-SNAREs and His6-tagged Q-SNAREs were mixed at 4 μM each in 400 μl RB500 (20 mM HEPES-NaOH, pH 7.4, 500 mM NaCl, 10% glycerol) containing 100 mM β-OG, incubated at 4°C for 1 h with gentle agitation, mixed with glutathione-Sepharose 4 Fast Flow beads (200 μl, 50% slurry; GE Healthcare, Parsippany, NJ, USA) equilibrated in the same buffer, and further incubated at 4°C for 1 h. The glutathione-Sepharose beads were isolated by centrifugation (2 min, 15,300 g, 4°C) and washed four times in 400 μl RB500 containing 100 mM β-OG. Bound GST-tagged R-SNAREs and His6-tagged Q-SNAREs were eluted at 100°C for 5 min with 2% SDS, followed by SDS-PAGE and Coomassie Blue staining. For the GST pull-down assays with the SM protein Sly1p, the glutathione-Sepharose beads were preincubated with QabcR-SNARE sets (GST-His6-tagged R-SNAREs and His-tagged Qabc-SNAREs, 4 μM for each SNARE) at 4°C for 2 h with gentle agitation and then washed twice in 400 μl RB500 containing 1% Triton X-100 instead of 100 mM β-OG. His6-tagged Sly1p (4.6 μM final) was added to the washed beads in 500 μl RB500 with 1% Triton X-100. After a 4°C incubation for 1 h with gentle shaking, the beads were further washed three times in 400 μl RB500 with 1% Triton X-100. Bound QabcR-SNAREs and Sly1p were eluted at 100°C for 5 min with 2% SDS and analyzed by SDS-PAGE and Coomassie Blue staining.
5
GST Pull-Down Assay for Protein Interactions
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The glutathione S‐transferase (GST) pull‐down assay was performed according to the previously described methods (Mehto et al, 2019a (link), 2019b ). Briefly, GST or GST‐RIPK2 or GST‐IRGM recombinant proteins were expressed in E. coli SoluBL21 (Amsbio), and the proteins were purified on Glutathione Sepharose 4 Fast‐Flow beads (GE Healthcare). The [35S] labeled‐ Myc‐NOD1, Myc‐NOD2, Myc‐RIPK2, Myc‐ULK1, Myc‐ATG16L1 or Myc‐Beclin‐1 proteins were in vitro translated using TnT T7–coupled reticulocyte lysate system (Promega). The GST proteins were incubated with [35S]‐labeled proteins in 200 μl of NETN‐E buffer (50 mmol/l Tris, pH 8.0, 100 mm NaCl, 6 mm EDTA, 0.5% NP‐40, and 1 mm dithiothreitol (DTT) supplemented with complete mini EDTA‐free protease inhibitor cocktail; Roche) for 2 h at 4°C. After incubation, the beads were washed five times with NETN‐E buffer, boiled with loading buffer, and subjected to SDS–PAGE. The gels were stained with coomassie blue and vacuum‐dried. The GST was detected by staining with coomassie blue stain, whereas the [35S]‐ labeled Myc‐tagged proteins were detected in PharosFX imager (Bio‐Rad Laboratories).
6
Recombinant Protein Expression and Purification
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Recombinant protein expression constructs were transformed into various E. coli strains for optimum expression (table S2). Protein samples used for EMSA studies were expressed in standard LB media. Proteins used for acquiring 15N HSQC NMR spectrum were expressed in M9 minimal media, with 15N-labeled ammonium chloride (15N, 98%+) (Cambridge Isotope Laboratories Inc.) as nitrogen source and d -glucose (Cambridge Isotope Laboratories Inc.) as carbon source. Protein expression was induced by isopropyl-β-d -thiogalactopyranoside. TrxA-His6–tagged proteins were purified with a His•bind resin (Millipore) according to the manufacturer’s protocol. GST-tagged proteins were purified with Glutathione Sepharose 4 Fast Flow beads (GE Healthcare) according to the manufacturer’s protocol. For protein samples used for acquiring the 15N HSQC NMR spectrum, the fusion tag was removed by 3C protease. Purified proteins were dialyzed in dialysis buffer [20 mM Hepes (pH 7.9), 100 mM KCl, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 15% glycerol, and 1 mM dithiothreitol (DTT)] at 4°C overnight.
7
GST Pulldown Assay for PRR Interactions
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GST pulldown assay was done as described previously (Mehto et al, 2019 ). GST‐IRGM recombinant protein was expressed in SoluBL21 (Amsbio) and purified on Glutathione Sepharose 4 Fast‐Flow beads (GE Healthcare). [35S]‐labeled Myc‐cGAS, Myc‐RIG‐I, and Myc‐TLR3 were cotranscribed/translated using the TnT T7‐coupled reticulocyte lysate system (Promega). The in vitro‐translated [35S]‐labeled proteins were then incubated with GST or GST‐IRGM in 250 μl of NETN‐E buffer (50 mM Tris pH 8.0, 100 mM NaCl, 6 mM EDTA, 6 mM EGTA, 0.5% NP‐40, and 1 mM dithiothreitol supplemented with Complete mini EDTA‐free protease inhibitor cocktail [Roche]) for 2 h at 4°C and then washed five times with 1 ml of NETN‐E buffer, boiled with loading buffer, and subjected to SDS–PAGE. The gel was stained with Coomassie Blue and vacuum‐dried. The GST‐IRGM was detected by staining with Coomassie Blue, whereas the [35S]‐labeled PRRs were detected in PharosFX imager (Bio‐Rad Laboratories).
8
Purification and Characterization of GST-Tagged Proteins
9
Histone H2B Binding and Phosphorylation Assays
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For H2B and CHMP4B binding assays, GST-CHMP4B (ag4544, Proteintech, Rosemont, IL, USA) was incubated overnight at room temperature with 500 ng of recombinant His-H2B (ag7811, Proteintech) or histone H2B (#14-491, Millipore) in buffer phosphate pH 7.5, 150 mM NaCl. GST-pulldown was performed by incubation for 2 h at 4 °C with Glutathione-Sepharose 4 Fast Flow beads (GE Healthcare, Buckinghamshire, UK) and three washes with buffer phosphate. Bound proteins were resolved by SDS-PAGE and analyzed by WB. For H2B phosphorylation, recombinant His-H2B was incubated with HIPK2 Kinase domain (kind gift of Dr. Linda Montemiglio), as an enzymatic source, in kinase buffer (Hepes 20 mM pH 7.5, 1 mM DTT, 10 mM MgCl2, and 1 mM EGTA) at 30 °C for 30 min in the presence of cold ATP or, as a control, of γ-32P-ATP (BLU502Z250UC, Perkin-Elmer, Waltham, MA, USA).
10
Pre-methylated GST-hnRNPK Purification
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cDNA encoding the full-length human hnRNPK fragment from pET23a-Trx-hnRNPK was subcloned into the EcoRI/XhoI sites of pGEX4T-1 to establish a GST-hnRNPK fusion protein. The GST-hnRNPK fragment with NcoI and XhoI sites were obtained through polymerase chain reaction (PCR) and further subcloned into the NcoI and SalI sites of the pET-DUET vector at multiple cloning site 1. The resulting control plasmid was named pET-DUET-GST-hnRNPK. In addition, cDNA encoding the full-length human PRMT1 fragment from pCDNA3HA2-PRMT1 (35 (link)) was subcloned into the NdeI/XhoI site of the pET-DUET vector at multiple cloning site 2. Moreover, the lipoprotein (lpp) promoter fragment, carrying the BsrGI and NdeI sites, was PCR amplified from pACYC184-lpp (36 (link)). The PRMT1 and lpp promoter fragments were annealed and subcloned into the pET-DUET-GST-hnRNPK to construct pET-Duet-GST-hnRNPK-lpp-PRMT1 to generate pre-methylated hnRNPK.
Escherichia coli BL21(DE3) cells harboring pETDUET-GST-hnRNPK or pETDUET-GST-hnRNPK-lpp-PRMT1 plasmids were cultured in LB medium. The expression of GST-hnRNPK or pre-methylated GST-hnRNPK was induced using 0.2 μM IPTG, and the recombinant proteins were purified using glutathione-Sepharose 4 Fast Flow beads (GE Healthcare Bio-Sciences, Uppsala, Sweden) according to the manufacturer's instructions.
Escherichia coli BL21(DE3) cells harboring pETDUET-GST-hnRNPK or pETDUET-GST-hnRNPK-lpp-PRMT1 plasmids were cultured in LB medium. The expression of GST-hnRNPK or pre-methylated GST-hnRNPK was induced using 0.2 μM IPTG, and the recombinant proteins were purified using glutathione-Sepharose 4 Fast Flow beads (GE Healthcare Bio-Sciences, Uppsala, Sweden) according to the manufacturer's instructions.
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