GST-RILP pulldown assays were performed as previously described in detail [31 (link),59 (link)]. In brief, freshly purified GST-RILP protein bound to GSH-Sepharose 4B beads (GE Healthcare) was incubated with extracts from transfected HEK293T cells (one 10 cm diameter dish per assay) in pulldown buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1% Triton X-100, and 1 mM PMSF), and incubated overnight in a rotary wheel at 4 °C. Beads were washed twice with ice-cold pulldown buffer, and bound protein eluted with 40 μL of 1× sample buffer/β-mercaptoethanol and heating for 4 min at 95 °C prior to SDS-PAGE.
Gsh sepharose 4b beads
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
GSH-Sepharose 4B beads are an affinity chromatography resin used for the purification of glutathione S-transferase (GST) fusion proteins. The beads consist of cross-linked agarose matrix covalently coupled with glutathione, which binds to the GST tag of the fusion protein. This allows for the selective capture and purification of the target protein from complex mixtures.
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Lab products found in correlation
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg secondary antibody, by Bio-Rad (1 mentions)
Hyperfilm ecl, by GE Healthcare (1 mentions)
Bradford based protein assay kit, by Bio-Rad (1 mentions)
Prescission protease, by GE Healthcare (1 mentions)
Tnt coupled reticulocyte lysate system, by Promega (1 mentions)
11 protocols using gsh sepharose 4b beads
1
GST-RILP Protein Pulldown Assay
2
Calmodulin Binding Assay Using GST Fusion
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Purified GST fusion proteins immobilized on GSH-Sepharose 4B beads (GE Healthcare) were equilibrated in pull-down buffer (25 mM Hepes, 120 mM KCl, 5 mM NaCl, pH 7.5) with either 100 μM CaCl2 or 5 mM EGTA. Calmodulin (10 μg) was added to the beads and incubated for 45 min at room temperature. After three washes, the proteins were recovered, separated by 15% SDS-PAGE, and transferred to Immobilon-P PVDF membrane (Merck Millipore) for Western blotting. The membrane was blocked with 5% nonfat milk in TBS-T (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.5), incubated with the primary antibody (monoclonal anti-CaM 05–173 from Merck Millipore, diluted 1:2000 in blocking buffer) O/N at 4°C, washed, and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad) diluted 1:5000 in blocking buffer. Antibody binding was detected using enhanced chemiluminiscence and ECL hyperfilm (GE Healthcare). GST-fusion proteins (input) were identified using an Anti-GST–Peroxidase Conjugate antibody (dilution 1:5000; A7340 Sigma). ImageJ software (Version WCIF, National Institutes of Health, USA) was used to quantify band intensities and calculate the relative CaM binding % (n = 3).
3
Purification and Pull-down of GST-fused Proteins
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We purified the GST-fused CaM or CaM1234 according to the previously published protocol [21 (link)] and used a Bradford-based protein assay kit (Bio-Rad, CA, USA) to estimate concentration. To pull down interacting proteins, we incubated GST-fused proteins or GST (1 mg) with GSH-Sepharose 4B beads (GE Healthcare, U.S.A.) following the protocol suggested by the manufacturer. We mixed the beads with cell lysates at 4°C overnight in a Binding Buffer (100 mM NaCl, 50 mM MgCl2, 10% Glycerol, 0.1% Nonidet P-40, 0.2% BSA, 25 mM HEPES, and 1 mM DTT) containing a Protease Inhibitor Cocktail (Set V, 1:100 dilution, CalBiochem, La Jolla, California, USA). The proteins bound to the beads were analyzed by SDS-PAGE followed by Western blot.
4
Rab GTPase Interaction Assay
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HEK cells were transfected with cDNA encoding GFP-Rab constructs (either wild-type or mutant forms) and lysed 24–48 h posttransduction in IP buffer containing 1 mM GTP. The cell lysate was centrifuged and the supernatant “precleared” by incubating the lysate with GSH Sepharose-4B beads (GE Healthcare) for 30 min at 4°C. After a brief centrifugation at 2000 × g, an aliquot of lysate containing 250 μg of total protein was mixed with 1 μg of purified TBC1D9B fusion protein and interacting complexes recovered by the addition of 25 μl of GSH beads and incubation for 3 h at 4°C on a rotator. The reactions were centrifuged for 1 min at 2000 × g, and the beads were washed five times with IP lysis buffer. The beads were resuspended in 25 μl of 2× Laemmli sample buffer, heated at 70°C for 10 min, the proteins resolved by SDS–PAGE, and Western blots probed with the indicated primary antibodies. When stated in the text, no MgCl2 was added to the IP buffer. To quantify the binding efficiency in the pull-down experiments, we quantified the bands detected in the immunoblots using ImageJ software. Values were normalized to total expression of each protein in the lysate and the corresponding amount of GST protein used in each experiment.
5
Affinity Purification of Stress Sensors
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Protein A Sepharose 4B beads (Zymed Invitrogen) and appropriate antisera (against IRE1 and PERK), or glutathione (GSH) Sepharose 4B beads (GE Healthcare) were equilibrated in lysis buffer. 20 μL beads per sample were added to lysates and left rotating for 1 hour at 4°C. The beads were then washed in lysis buffer and residual liquid was removed using a syringe. The protein from the beads was eluted in SDS sample buffer containing 20 mM DTT or 20 mM NEM (for non reducing gels).
6
Purification and Cleavage of Essential Cytoskeletal Proteins
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GST-tagged CLIC4 (WT and C35A) was expressed in BL21 Escherichia coli, and CLIC4 proteins were purified using GSH-Sepharose ® 4B beads (GE Healthcare) and gel filtration. The GST tag was removed with PreScission Protease (GE Healthcare). Expression, purification, and cleavage of human profilin-1 were performed as described previously (18 (link)). Expression and purification of mDia2 (FH1–FH2 domain) were previously described (34 (link)), and cleavage was done as described for profilin-1 (18 (link)). Expression and purification of Rho–GTPase binding domains were previously described (50 (link)).
7
Purification of GST-VBP1 Fusion Protein
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Escherichia coli BL21 competent cells were transformed with GST or GST-VBP1 and then induced with 0.1 mm isopropyl β-d -thiogalactopyranoside at 37 °C for 5 h. The cells were collected by centrifugation, washed once with ice-cold PBS, and then lysed by sonication in lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 10% glycerol, and 1% Triton X-100) on ice. The GST or GST–VBP1 extract was then mixed with GSH Sepharose 4B Beads (GE Healthcare) overnight on a rotator at 4 °C. These mixtures were centrifuged, and the precipitates were diluted with an equal amount of indicated cell lysates of HEK293T and rotated for 2 h at 4 °C. After extensive washing with the lysis buffer, the precipitated proteins were eluted using SDS/PAGE. The bound proteins were detected by indicated antibodies or Coomassie Blue staining.
8
Purification and Pulldown of GST-Fusion Proteins
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GST fusion protein expression, protein purification and GST-pulldown assays were performed essentially as described.33 (link) In brief, GST fusion proteins were expressed in the E.coli BL21 strain and purified using glutathione (GSH) sepharose 4B beads (GE Healthcare, GE Healthcare UK Limited, Buckinghamshire, England). 0.2–0.4 μg of bead-coupled GST fusion proteins were used for the pulldowns. GST fusion proteins were incubated with proteins generated by in vitro translation using the TNT Coupled Reticulocyte Lysate System (Promega Corporation, Madison, WI, USA). GST-SIRT1, 35S-Flag-HIPK2 pull down was performed in 20 mM Tris-HCl, pH7.4, 0.05% NP40, whereas GST-HIPK2, 35S-Flag-SIRT1 pull down in 1 × PBS, 0.05% NP40.
9
Pulldown Assay for RUNX3-MYC Interaction
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pGEX4T-1 plasmids containing glutathione‐S‐transferase (GST) and GST-tagged Runt domain (RUNX3 aa 49–187) were expressed and purified from E. coli RosettaTM DE3 cells after induction with 0.1 mM IPTG for 3 h at 37 °C. The GST proteins were purified by glutathione (GSH) Sepharose 4B beads (GE Healthcare). For the pulldown assay, GST proteins bound to GSH beads were incubated with 5 µg of recombinant MYC (RayBiotech). After copious washes, the precipitated proteins were resolved by SDS-PAGE and analyzed by immunoblot using anti-MYC (1:1000) and anti-GST (1:200) antibodies.
10
Purification of GST-tagged proteins from E. coli
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E. coli cells harboring the pGEX4T1 GST-Tom20 cytosolic domain, pGEX4T1 GST-Tom70 cytosolic domain, or pGEX4T1-GST plasmid (Hoseini et al., 2016 (link)) were grown to an OD600 of 0.8 at 37°C, and 0.5 mM IPTG was added. After incubation for 16 h, 50 ml of the culture was harvested. The cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1% NP-40, and 1 mM β-mercaptoethanol) and incubated with 1 mg/ml lysozyme for 30 min at room temperature. After one step of freeze thawing and 15 cycles of sonification for 1 s at 60% duty level, the lysate was cleared at 25,000 g for 5 min at 4°C. The supernatant was incubated with GSH Sepharose 4B beads (GE Healthcare) and washed 3× with PBS. Bound GST fusion proteins were eluted by incubation with elution buffer (50 mM Tris-HCl, pH 8.0, 25 mM GSH, and 1 mM DTT) for 30 min at 4°C. Purification efficiency was analyzed by SDS-PAGE and Coomassie brilliant blue staining.
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