The Exosc3 open reading frame encoding the mouse EXOSC3 protein was cloned into pGEX-6P-2 plasmid (GE Healthcare Life Sciences [now Cytiva]) to create an N terminally GST-tagged EXOSC3 construct. Recombinant GST-EXOSC3 was expressed in Escherichia coli Rosetta 2 (DE3). The GST fusion protein was purified by affinity chromatography on GSH-Sepharose (GE Healthcare Life Sciences [now Cytiva]) in 300 mM NaCl, 50 mM Hepes/NaOH (pH 7.5), 5% glycerol and 2 mM beta-mercapto-ethanol. The fusion protein was eluted by addition of 30 mM reduced GSH. The GST tag was removed using PreScission protease (GE Healthcare Life Sciences [now Cytiva]), and the EXOSC3 protein was further purified by collecting the flowthrough of a second affinity chromatography on GSH-Sepharose resin. The untagged protein was further purified on a Superdex200 size-exclusion chromatography column equilibrated in 150 mM NaCl, 30 mM Hepes/NaOH (pH 7.5), 5% glycerol. Expression and purification of both recombinant GST-EXOSC3 and untagged EXOSC3 were confirmed by SDS-PAGE, followed by Coomassie staining. The purified untagged protein was used as an immunogen to raise rabbit polyclonal antibodies by Josman, LLC. Sera containing anti-EXOSC3 antibodies was collected 21 days after immunization and used directly for immunoblotting, immunoprecipitation, and immunofluorescence.
Gsh sepharose
Manufactured by GE Healthcare
Sourced in Italy
GSH-Sepharose is a chromatography resin composed of glutathione (GSH) immobilized on Sepharose beads. It is used for the purification of glutathione S-transferase (GST) fusion proteins through affinity chromatography.
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Lab products found in correlation
Prescission protease, by GE Healthcare (4 mentions)
Ni2 nitrilotriacetic acid resin, by Qiagen (2 mentions)
Ni2 sepharose, by GE Healthcare (2 mentions)
Instantblue, by Abcam (1 mentions)
Anti β actin ac 15, by Merck Group (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Pgex 4t 1 vector, by GE Healthcare (1 mentions)
Casein, by Carl Roth (1 mentions)
Cacl2, by Merck Group (1 mentions)
Isopropyl β d thiogalactopyranoside, by Merck Group (1 mentions)
B per complete bacterial protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Pegfp n1, by Addgene (1 mentions)
Pgex 6p 2 plasmid, by GE Healthcare (1 mentions)
Tryptone, by Merck Group (1 mentions)
Yeast extract, by Merck Group (1 mentions)
Lympholyte h, by Euroclone (1 mentions)
Sharpmass 6 mw marker, by Euroclone (1 mentions)
L glutamine 100x 200 mm, by Euroclone (1 mentions)
Protease inhibitor cocktail 100x, by Cell Signaling Technology (1 mentions)
17 protocols using gsh sepharose
1
Recombinant EXOSC3 Protein Production
2
Recombinant SntA Protein Expression
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The constructs bearing the sequences coding for the SntA core and its mutants contain the cloned coding sequences in frame with those of the PreScission protease cut sequence and the glutathione S-transferase (GST) label. The recombinant proteins can be expressed from the isopropyl ß-D-1-thiogalactopyranoside-inducible tac promoter of the pGEX-6P-3 vector as GST fusions that can be cut with PreScission protease (GE Healthcare Life Sciences, available from VWR International Eurolab SLU, Llinars del Vallès, Spain) to yield the desired SntA protein with a GPLGS N-terminal extension. The expression and purification of the recombinant proteins were performed as described (López-Villamizar et al., 2016 (link)). In brief: each expression construct was used to transform BL-21 cells; ampicillin-resistant colonies were selected, cultured in suspension, induced by isopropyl ß-D-1-thiogalactopyranoside, and collected by centrifugation. The cells were resuspended in the presence of a protease inhibitor cocktail, lysed by sonication, and the lysis supernatant was taken for purification of the recombinant proteins. Purification was accomplished by affinity chromatography on GSH-Sepharose (GE Healthcare Life Sciences, available from VWR International Eurolab SLU, Llinars del Vallès, Spain), followed by separation from the GST label by specific proteolysis with PreScission.
3
Recombinant Tyrosine Hydroxylase Protein Purification
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Recombinant proteins with His or GST tag were expressed in Rosetta (DE3) pLys Escherichia coli (Novagen, Bilerica, MA). Cultures were grown until linear phase (0.6 OD) and expression of proteins were induced by 250 μM IPTG at 18 °C for 20 h. As iron is essential for TH activity, to maintain TH activity 2% glucose and 1 mM FeSO4 together were added when inducing the expression of recombinant TH as reported29 (link). The culture was then harvested and recombinant proteins were purified by Ni2+-nitrilotriacetic acid resin (Qiagen) or GSH-Sepharose (GE Healthcare, Piscataway, NJ) according to the manufacturer’s protocol, respectively. The obtained proteins were then dialyzed in PBS.
For the generation of anti-TH antibody, purified his-tagged TH recombinant protein in PBS was mixed with equal volume of Freund’s adjuvant forming an emulsion. The mixture was then used to immunize a New Zealand white rabbit and the Anti-TH antibody was purified from serum with GST-tagged TH conjugated to CNBr activated Sepharose affinity purification (GE Healthcare, Piscataway, NJ). The specificity of the antibodies was verified by Western blot analysis.
For the generation of anti-TH antibody, purified his-tagged TH recombinant protein in PBS was mixed with equal volume of Freund’s adjuvant forming an emulsion. The mixture was then used to immunize a New Zealand white rabbit and the Anti-TH antibody was purified from serum with GST-tagged TH conjugated to CNBr activated Sepharose affinity purification (GE Healthcare, Piscataway, NJ). The specificity of the antibodies was verified by Western blot analysis.
4
Bait-Prey Protein Interaction Assay
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For interaction studies the respective combination of proteins 80 μM of bait protein (GST-Mtr4SA, residues 638–842, wild-type and mutants) was preincubated with 100 μM of prey (Z-tagged Nop53, residues 48–99) in a buffer containing 20 mM Hepes/NaOH pH 7.5, 150 mM NaCl, 5 mM DTT, and 0.05% (v/v) NP-40 for 1 h at 0°C. Then the sample was incubated with GSH Sepharose (GE Healthcare) for 2 h at 4°C, washed three times with the same buffer, and eluted in 20 mM Tris/HCl pH 7.5, 150 mM NaCl, 2 mM DTT, 0.01% (v/v) NP-40 and 30 mM reduced gluthathione. Input and pull-down fractions were analyzed on denaturing 16% SDS–PAGE and visualized by staining with Instant Blue (Expedeon).
5
Purification of GST-Fusion Proteins
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To express GST-fused proteins or peptides, BL21 E. coli cells transformed with appropriate plasmids were grown at 37 °C in Luria–Bertani medium with the addition of antibioticum for plasmid selection to 0.6 OD600. Afterward, cells were incubated overnight at 16 °C in the presence of 1 mm isopropyl 1-thio-β-d -galactopyranoside and 2% ethanol. After harvesting, cells were resuspended in PBS containing 5 mm EDTA and protease inhibitors and disrupted by sonication. Debris was removed by centrifugation (two times at 16,000 × g for 13 min). 1 mg/ml BSA and GSH-Sepharose (GE Healthcare) were added, and the samples were rotated for 1 h at 4 °C. 20 μl of GSH-Sepharose were loaded with the extract of 120 OD600E. coli cells. Afterward, the beads were washed three times in PBS with the addition of 1% Triton X-100, 200 mm NaCl, 1 mg/ml BSA, and protease inhibitors. After washing, the beads were rotated with either yeast or HEK293 cell extract overnight. The beads were washed three times in incubation buffer and eluted with sample buffer. Yeast and HEK293 cell extracts were prepared as for coimmunoprecipitation experiments, and 1 mg/ml BSA was added before incubation with Sepharose. Extract of 3.4 OD600 yeast cells or extract of HEK293 cells derived from one well of a 6-well plate was loaded onto the beads.
6
Purification and Antibody Generation for NME7
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Recombinant proteins containing a hexahistidine (His6) or glutathione S-transferase (GST) tag were expressed in Escherichia coli BL21 (DE3) and then isolated using Ni2+-nitrilotriacetic acid resin (Qiagen, Valencia, CA) or GSH-Sepharose (GE Healthcare, Chalfont St. Giles, Buckinghamshire, United Kingdom), respectively. After elution, the proteins were dialyzed in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM ethylene glycol tetraacetic acid (EGTA), and 10% glycerol and then stored at −80°C.
Two anti-NME7 antisera were generated by immunizing rabbits with NME7 1–140 and full-length NME7 prepared as His6-tagged proteins. The sera obtained were named sera 651 and 673, respectively. Both antibodies were purified using respective antigens in fusion with GST and immobilized on polyvinylidene fluoride membranes. The two antibodies gave similar results in experiments, and most of the data presented here were collected using antibody 651. The production of the following antibodies has been described previously: rabbit anti-CDK5RAP2, anti-GCP2, anti-GCP3, anti-GCP4, anti-GCP5, and anti-GCP6 (Fong et al., 2008 (link); Choi et al., 2010 (link)). These mouse monoclonal antibodies were purchased from Sigma (St. Louis, MO): anti-FLAG (M2), anti–γ-tubulin (GTU-88), anti–α-tubulin (DM1A), and anti–β-actin (AC-15).
Two anti-NME7 antisera were generated by immunizing rabbits with NME7 1–140 and full-length NME7 prepared as His6-tagged proteins. The sera obtained were named sera 651 and 673, respectively. Both antibodies were purified using respective antigens in fusion with GST and immobilized on polyvinylidene fluoride membranes. The two antibodies gave similar results in experiments, and most of the data presented here were collected using antibody 651. The production of the following antibodies has been described previously: rabbit anti-CDK5RAP2, anti-GCP2, anti-GCP3, anti-GCP4, anti-GCP5, and anti-GCP6 (Fong et al., 2008 (link); Choi et al., 2010 (link)). These mouse monoclonal antibodies were purchased from Sigma (St. Louis, MO): anti-FLAG (M2), anti–γ-tubulin (GTU-88), anti–α-tubulin (DM1A), and anti–β-actin (AC-15).
7
Protein Interaction Characterization Protocol
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Full length versions and deletion mutants of human MITF, MYC, and CDK7 were expressed as GST-fusion proteins in AVB100 bacteria. For MITF and MYC, the GST-tag was removed by digestion with Prescission protease (GE Healthcare, 27-0843-01). For GST pull down, 12.5 µg of GST-CDK7 were bound to 10 µl of GSH-sepharose (GE Healthcare, 17075601) in the presence of protease inhibitors for 1 h at RT. Subsequently, beads were washed (2×) with PBS, blocked with 1% BSA in PBS for 30 min at RT, incubated for 1.5 h at 4 °C with 3–6 µg GST-free MITF and MYC, respectively, washed with PBS and eluted with 20 mM GSH (Sigma, 4251) in 100 mM Tris-HCl (pH 8.0) for Western blot analysis; free GST served as binding control.
8
Purification of Nedd8-related Proteins
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The following purified proteins were previously described in Kelsall et al., (2013 (link)): Nedd8 and the APPBP1–UBA3 heterodimer (Ohta et al., 2013 (link)); DAC–TEV–Cul3–RBX1 and KLHL3 (Schumacher et al., 2015 (link)); GST–CAND1. DCNL1, 2, 3, 4 and 5 were expressed with His6 tags in BL21 and purified by Ni2+–Sepharose (GE Healthcare). GST–DCNL1 and GST–DCNL2 were purified by GSH–Sepharose (GE Healthcare).
9
Overexpression and Purification of GST- and His-Tagged Proteins in E. coli
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Escherichia coli BL-21 cells carrying plasmids expressing the various GST- or 6×His-tagged fusion proteins were grown in 125 ml of LB medium (0.5% yeast extract, 1% tryptone, 1% NaCl) to a late exponential phase. After induction of protein expression with the addition of 0.5 mM isopropyl-β-d -thiogalactopyranoside, cells were grown at 37°C or 20°C for 4 h or 16 h. Bacteria were harvested, resuspended in 4 ml of lysis buffer (phosphate-buffered saline [PBS] [137 mM NaCl, 10 mM phosphate [pH 7.4], 2.7 mM KCl] containing 5 mM dithiothreitol [DTT], 1 mg/ml lysozyme, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10% glycerol, 1% Triton X-100, and complete protease inhibitor [Roche, Basel Switzerland]), and lysed by two 10-s rounds of sonication using a Branson sonicator (Brandon, Danbury, CT). The bacterial lysates were cleared by centrifugation at 13,000 rpm for 10 min at 4°C. The concentration of 6×His-tagged proteins was measured using a bicinchoninic acid (BCA) kit (Promega, Madison, WI). Proteins were not further purified, and bacterial lysates were stored at −80°C until used. For purification of GST fusion proteins, lysates were incubated with 125 μl of GSH-Sepharose (GE Healthcare), which had been prewashed in PBS on a rotatory wheel at 4°C for 2 h.
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10
Expression and Purification of p22phox Proteins
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p22phox full-length (FL) or C-terminal (CT) coding sequence (CDS) was subcloned from the DsRed-p22phox construct into the pGEX-4T-1 vector (GE healthcare, Danderyd, Sweden), and the resulting constructs, GST-p22phox FL and GST-p22phox CT, were transformed into the E. coliStbl3 strain (Thermo Fisher Scientific, Waltham, MA, USA). After growing the bacterial cultures overnight at 37 °C, recombinant protein expression was induced by the addition of 0.2 mM IPTG to the culture at room temperature overnight. The bacterial cells were collected by centrifugation at 4 °C and resuspended in 10 mL PBST (1% Triton X-100) buffer. Bacterial lysates were obtained by sonication on ice and then removal of insolubl
e pellets by centrifugation. To purify the recombinant proteins, 1 mL of glutathione (GSH) sepharose (GE healthcare, Danderyd, Sweden) was added into the bacterial lysates, and the GSH sepharose mixture was incubated overnight at 4 °C. The sepharose beads were washed in the PBST buffer, and the GST-tagged recombinant proteins were eluted by fresh 10 mM reduced GSH (Sigma, St. Louis, MO, USA). The resulting GST-p22phox FL and GST-p22phox CT proteins were verified by Western blot analysis.
e pellets by centrifugation. To purify the recombinant proteins, 1 mL of glutathione (GSH) sepharose (GE healthcare, Danderyd, Sweden) was added into the bacterial lysates, and the GSH sepharose mixture was incubated overnight at 4 °C. The sepharose beads were washed in the PBST buffer, and the GST-tagged recombinant proteins were eluted by fresh 10 mM reduced GSH (Sigma, St. Louis, MO, USA). The resulting GST-p22phox FL and GST-p22phox CT proteins were verified by Western blot analysis.
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