GST fusion proteins were expressed in E. coli BL21 (DE3) and purified on glutathione–sepharose beads (Amersham Biosciences, Little Chalfont, UK) following standard procedures. Briefly, Glutathione S-transferase (GST)-tagged ITCH protein and inactivated ITCH mutant (C830A) were expressed in bacteria. E. coli BL21 CodonPlus(DE3)-RIL cells (Stratagene, La Jolla, CA, USA) were transformed with either wild type or mutant constructs prepared in the expression vector pGEX-6P1 (Amersham Biosciences). Saturated cultures were prepared by inoculation of LB medium containing ampicillin (LB/amp) with growth overnight at 37 °C. For expression, overnight cultures were diluted 1/100 in LB/amp at 37 °C until they reached an OD of 0.40. At this point the temperature was reduced to 15 °C and IPTG (50 μM final concentration) was added to induce expression for 3–4 h with shaking at 250 r.p.m. Cell lystates were prepared and the GST fusion proteins were purified on glutathione–sepharose beads (Amersham Biosciences) using standard procedures.
Glutathione sepharose beads
Manufactured by Cytiva
Sourced in United States, United Kingdom, Sweden
Glutathione-Sepharose beads are a chromatography medium composed of glutathione, a tripeptide, covalently coupled to Sepharose beads. They are designed for the affinity purification of proteins that contain a glutathione-S-transferase (GST) tag.
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Lab products found in correlation
Complete protease inhibitor cocktail, by Roche (4 mentions)
Ni nta agarose bead, by Qiagen (3 mentions)
Pqe30, by Qiagen (3 mentions)
Anti gst, by Abcam (2 mentions)
Gst tag, by Merck Group (2 mentions)
His tag antibody, by Merck Group (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Fluorescent ubiquitin, by Thermo Fisher Scientific (2 mentions)
Mammalian e1, by Enzo Life Sciences (2 mentions)
His p53, by Enzo Life Sciences (2 mentions)
Denaturing loading buffer, by Thermo Fisher Scientific (2 mentions)
Ni nitrilotriacetic acid agarose beads, by Qiagen (2 mentions)
Prescission protease, by GE Healthcare (2 mentions)
His tag antibody, by Abcam (2 mentions)
Tnt t7 quick coupled transcription translation system, by Promega (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pegfp n3, by Takara Bio (2 mentions)
Pcmv5 myc, by Qiagen (2 mentions)
Pcdna3.1 v5 his, by Thermo Fisher Scientific (2 mentions)
Pclxsn gfp, by Takara Bio (2 mentions)
101 protocols using glutathione sepharose beads
1
Purification of GST-tagged ITCH Protein
2
Purification and Interaction Analysis of Viral Proteins
3
GST-tagged AP-1 Complex Pull-down
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For AP-1–ARF1 pull-down, 30 μg GST-tagged AP-1 complex, 10 μg ARF1 and 10 μg LBD-STING were mixed together or individually in 40 μl pull-down buffer (PBS supplemented with 2 mM MgCl2, 1 mM GTP and 2 mM TCEP). The mixture was incubated overnight on ice. Thirty microlitres of glutathione sepharose beads (Cytiva) were incubated with the mixture for 30 min at 4 °C. Excess proteins were washed off the beads using 200 μl pull-down buffer each time for four times. Twenty microlitres of 5× SDS loading buffer was added to the resin and the mixture was boiled for 5 min. The samples were then centrifuged briefly. Five microlitres of supernatant was analysed by SDS–PAGE. The protein bands were visualized by Coomassie blue staining. For AP-1 ΔμCTD pull-down, 30 μg GST-tagged AP-1 ΔμCTD complex and 10 μg LBD-STING were mixed together or individually in 40 μl PBS supplemented with 2 mM TCEP. The mixture was incubated overnight on ice. Thirty microlitres of glutathione sepharose beads (Cytiva) was incubated with the mixture for 30 min at 4 °C. Excess proteins were washed off the beads using 200 μl PBS each time for four times. Twenty microlitres of 5× SDS loading buffer was added to the resin and the mixture was boiled for 5 min. The samples were then centrifuged briefly. Five microlitres of supernatant was analysed by SDS–PAGE. The protein bands were visualized by Coomassie blue staining.
4
Characterization of GST- and His-tagged Proteins
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The full-length ORFs of RSMV M or OAZ1 of R. dorsalis were cloned into the pGEX-3x vector for expressing the GST fusion proteins (GST-M or GST-OAZ1). The full-length ORFs of OAZ1 or ODC1 of R. dorsalis were cloned into the pDEST17 vector for expressing the His fusion proteins (His-OAZ1 or His-ODC1). The recombinant proteins GST-M, GST-OAZ1, GST, His-OAZ1 and His-ODC1 were individually expressed in the Escherichia coli strain Rosetta. The GST-fused proteins were incubated with glutathione-Sepharose beads (Amersham) for 4 h at 4 °C, and the His-fused proteins were subsequently added to the beads and incubated for 2 h at 4 °C. After being centrifuged and washed, the bead-bound proteins were separated by SDS-PAGE and detected using antibodies against GST and His by western blot assays.
5
GST Fusion Protein Pulldown Assay
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GST fusion proteins were immobilized on glutathione–sepharose beads (Amersham Biosciences, USA). The beads were washed using pull-down buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mM DTT, 10% glycerol, 1 mM EDTA, 2.5 mM MgCl2, and 1 μg/mL leupeptin). The beads were incubated with recombinant protein tagged with His for two hours before being washed five times with binding buffer. Finally, the beads were resuspended in a sample buffer and the bound proteins were subjected to SDS-PAGE and Western blot analysis.
6
Quantifying Rho Activation in Cells
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Rho activation in cultured cells was assessed as follows30 (link): After serum starvation for 3 h, the cells were treated as indicated and lysed on ice in a buffer containing 20 mM Hepes, pH 7.4, 0.1 M NaCl, 1% Triton X-100, 10 mM EGTA, 40 mM β-glycerophosphate, 20 mM MgCl2, 1 mM Na3VO4, 1 mM dithiothreitol, 10 ug ml−1 aprotinin, 10 ug ml−1 leupeptin and 1 mM phenylmethylsulfonyl fluoride. The lysates were incubated with GST-rhotekin-Rho binding domain previously bound to glutathione-Sepharose beads (Amersham Biosciences) and washed three times with lysis buffer. Associated GTP-bound forms of Rho were released with SDS/PAGE loading buffer and analyzed by western blot analysis using a monoclonal antibody against RhoA, as described above.
7
Investigating TLR4-HIF-1α Interactions
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GST pull‐down assay was implemented to identify the interactions between TLR4 and HIF‐1α. For obtaining purified proteins, sequences encoding TLR4 were cloned into vector pEGX‐6P‐1 that contained open reading frame of GST tag, and sequences encoding HIF‐1α were cloned into vector pET22b that contained open reading frame of 6 × His tag. All the vectors were expressed in Escherichia coli BL21 and purified. Then, glutathione‐sepharose beads (Amersham Pharmacia Biotech, Shanghai, China) were incubated with purified GST‐fused proteins in a rotating incubator at 4°C for 12 hours. Then, the beads were collected and washed for 3 times, after which input proteins were incubated with the beads in a rotating incubator at 4°C for 3 hours. The supernatant was removed and beads were collected and washed, and target proteins were eluted and detected using Western blotting.
8
Quantifying Rho Activation in Cells
9
Purification and Interaction of GST-RIDα Fusion Proteins
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GST fusion proteins with RIDα cytoplasmic tail peptide fragments were described previously in [52 (link)]. Fusion proteins were purified from BL21 competent E. coli using the Inclusion Body Solubilization Reagent from Thermo Scientific (catalog number 78115), according to the manufacturer’s instructions. Inclusion body lysates were incubated with glutathione-Sepharose beads (Amersham-Pharmacia, catalog number 17075601) overnight at 4°C with rotation followed by three washes with a solution of 50 mM Tris (pH 7.4), 10 mM MgCl2, 0.15 M NaCl, and 1% Triton X-100. Beads with attached fusion proteins were incubated with whole cell lysates prepared from CHO cells using IP buffer. Beads were washed four times with IP lysis buffer, solubilized with sample buffer, resolved by SDS-PAGE, and immunoblotted with Alix antibody to detect bound proteins.
10
Rab7 and WDR91 Protein Interactions
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Recombinant GST-Rab7, GST-Rab7(T22N), and GST-Rab7(Q67L) proteins were expressed in BL21(DE3) bacterial cells and purified with glutathione–Sepharose beads (Amersham) according to the instructions provided by the supplier. 35S-labeled WDR91, WDR91(1–405), and WDR91(392–747) proteins were prepared by in vitro translation. Purified GST-tagged proteins (2.5 µg of each) were immobilized on glutathione–Sepharose beads and incubated with 35S-labeled WDR91, WDR91(1–405), or WDR91(406–747) in binding buffer (50 mM Tris-HCl, pH 8.8, 150 mM NaCl, 5 mM MgCl, 10 mM DTT, 0.05% NP-40, 1 mM PMSF, and protease inhibitor cocktail) at 4°C for 2 h. To examine the interactions of WDR91 with constitutively active (GTP) or dominant-negative (GDP) forms of Rab7, GST-Rab7 proteins immobilized on glutathione–Sepharose beads were first incubated in binding buffer supplemented with 10 mM GTP or 10 mM GDP at 37°C for 1 h and then incubated with 35S-labeled WDR91, WDR91(1–405), or WDR91(406–747) at 4°C for 2 h in binding buffer supplemented with 10 mM GTP or GDP. After extensive washing with binding buffer, bound proteins were resolved on SDS-PAGE and visualized by autoradiography.
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