The C85K mutation was inserted in pGEX6p, UbcH5c; the protein was expressed in E. coli with an overnight induction at 16 °C with 0.2 mM IPTG. After lysis in buffer containing 50 mM Hepes (pH 8), 150 mM NaCl, 0.5 mM TCEP the protein was purified using GSH beads (GE Healthcare) and eluted with 50 mM glutathione. The tag was cleaved by GST-3c overnight in dialysis with lysis buffer. After removal of the protease, the uncleaved protein and the GST with GSH beads, the protein was loaded into a S75 column in 20 mM Hepes (pH 8.0), 150 mM NaCl and 1 mM TCEP. The fractions containing the protein were concentrated and stored at −80 °C. The mutant E2 was loaded in a reaction containing 200 μM UbcH5c C85K, 200 μM ubiquitin, 1 μM Uba1 and 50 mM Tris (pH 9.5), 150 mM NaCl, 0.8 mM TCEP, 3 mM ATP and 5 mM MgCl2 (ref. 27 (link)). The reaction was incubated at 35 °C for ~20 h. The E2~Ub complex was purified on a S75 gel-filtration column and the fractions containing only the loaded E2 were concentrated and used for the gel-shift analysis shown in Fig. 4d,e .
Gsh beads
Manufactured by GE Healthcare
Sourced in United States
GSH beads are a type of lab equipment used for the purification and immobilization of glutathione (GSH) and GSH-conjugated proteins. They are made of agarose beads with immobilized glutathione, which can be used to capture and isolate proteins and other molecules that have an affinity for GSH.
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Lab products found in correlation
Cycloheximide (chx), by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti actin, by Proteintech (1 mentions)
Anti myc, by Thermo Fisher Scientific (1 mentions)
Anti ubi, by Santa Cruz Biotechnology (1 mentions)
Anti n cadherin, by Thermo Fisher Scientific (1 mentions)
Normal igg, by Santa Cruz Biotechnology (1 mentions)
Anti snail, by Cell Signaling Technology (1 mentions)
Mms 101p, by Fortrea (1 mentions)
Anti ha, by Fortrea (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Phosphor screen, by GE Healthcare (1 mentions)
Tnt quick coupled transcription translation kit, by Promega (1 mentions)
Complete protease inhibitor cocktail, by Merck Group (1 mentions)
Fla 5100 scanner, by Fujifilm (1 mentions)
Gsh beads, by Merck Group (1 mentions)
Adomet, by Merck Group (1 mentions)
Biomol green kit, by Enzo Life Sciences (1 mentions)
Complete proteinase inhibitor cocktail, by Roche (1 mentions)
15 protocols using gsh beads
1
Purification and Characterization of C85K UbcH5c
2
USP37 Regulation via Mutagenesis and Antibody Detection
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The human USP37 cDNA was subcloned into pcDNA3.1-Flag vector or PCDH vector and USP37-C350S was made using PCR-based site-directed mutagenesis method. All other constructs were generated using standard molecular cloning methods and were confirmed by DNA sequencing. Antibodies were commercially purchased: anti-USP37 (rabbit, 18465-1-AP, Proteintech), anti-Snail (rabbit, 3879, Cell Signaling), anti-Ubi (mouse, sc-8017, Santa Cruz), anti-actin (mouse, 60008-1-lg, Proteintech), anti-N-cadherin (mouse, 33-3900, Invitrogen), anti-HA (mouse, MMS-101P, Covance), anti-Flag (mouse, F3165, Sigma), anti-Flag (rabbit, F7425, Sigma), anti-Myc (mouse, 13-2500, Invitrogen), anti-GAPDH (mouse, 60004-1-Ig, Proteintech), normal IgG (rabbit, sc-2027, Santa Cruz), and GSH beads (GE). MG132 and cycloheximide (CHX) were obtained from Sigma.
3
GST Pull-down Assay for Cnot3 Interactions
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To perform the GST pull down with GST-Aurora B and GST-ERK1 with Cnot3 and the deletion mutants of Cnot3, 0.5 μg of pCDNA-Cnot3, pCDNA-Cnot3 Δ1-200, pCDNA-Cnot3 Δ651-700, pCDNA-Cnot3 Δ701-751, and pCDNA-Cnot3 Δ651-751 were in vitro transcribed/translated using a TNT Quick Coupled Transcription/Translation kit (Promega, USA) according to the manufacturer’s instructions using 10 μCi of 35S-methionine to radiolabel the proteins. GST or GST-Aurora B or GST-ERK (1 μg) was added to the GSH beads (GE Healthcare, USA) in binding buffer (50 mM Tris-Cl, pH 8.0, 150 mM monopotassium glutamate, 1 mM EDTA, 0.1% Igepal CAL630, 5% glycerol, 0.2% BSA), supplemented with Complete protease inhibitor cocktail (Merck, USA), and incubated for 2 h at 4°C. The beads were then washed and 5 μl of the in vitro transcribed/translated CNOT3 was incubated with the beads overnight at 4°C. The beads were then washed with the binding buffer, and the proteins were eluted by boiling in loading buffer. The eluted proteins were subjected to SDS–PAGE, stained, dried for 1 h at 80°C, and exposed overnight to Phosphor screen in a cassette (GE Healthcare, USA). Images were captured in a Fujifilm FLA 5100 scanner (Japan) using Fujifilm FLA-5000 software.
4
Analyzing G3BP1-eIF4E Binding and Methylation
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To detect the in vitro binding between G3BP1 and eIF4E, the purified recombinant proteins His-G3BP1 (2 μg, Novus) and GST-eIF4E (4 μg, Cayman) were mixed in Tris buffer containing various proteinase inhibitors and rotated overnight at 4 °C. GSH beads (50 μl, GE HealthCare) were added to the mixture and followed by 2 h rotation at 4 °C. The beads were washed three times with Tris buffer containing proteinase inhibitors and eluted with 2.5X sample buffer with 100 μM DTT. For the in vitro methylation of G3BP1, the purified proteins His-G3BP1 (2 μg), GST-eIF4E (4 μg), PRMT1 (4 μg, NKMAX), and AdoMet (80 μM, Sigma) were mixed in DPBS at 30 °C for 1 h, followed by binding assay by adding the reaction mix and GSH beads (50 ul) into D-PBS with proteinase inhibitors and rotated for 2 h in 4 °C as described above. After elution, samples were boiled at 100 °C for 5 min before SDS-PAGE and Western blot analysis.
5
Ebolavirus VP40 Binding Assay
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GST alone and GST-BAG3 WW domain fusion protein were expressed in BL-21 cells and subsequently conjugated to glutathione (GSH) beads (GE HEALTHCARE). HEK293T cells were transfected with eVP40-WT, eVP40-ΔPT/PY or flag-tagged-mVP40, respectively. At 24 hours after transfection, the cell extracts were incubated with the GSH beads described above at 4°C for 4 hours with continuous rotating. The proteins complexes were pulled down with beads via centrifugation. The rabbit eVP40 antiserum (PROSCI), mouse anti-flag monoclonal antibody (SIGMA), mouse anti-GST monoclonal antibody (SIGMA) were used to detect eVP40-WT, eVP40-ΔPT/PY, mVP40, GST, or GST-BAG3 WW proteins in input and pulldown samples by Western blotting.
6
Purification and Assay of AAA+ ATPase Modules
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The first and second AAA+ ATPase modules that are fused with GST were purified using GSH beads (GE Healthcare, USA) as previously described14 (link). ATPase activity was measured by malachite green assay using BIOMOL Green kit (Enzo Life Sciences, USA) as previously described14 (link)44 (link).
7
Purification and Analysis of LBR Proteins
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LBR211–EGFP, LBR211-all-A–EGFP, LBR211-all-D–EGFP, EGFP–NLS or EGFP was expressed in BL-21 bacteria. The recombinant proteins were induced by the addition of 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Nacalai Tesque), followed by 3 h of culture at 37°C. The cells were harvested, lysed by sonication in lysis buffer (50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 0.1% Triton X-100, 1 mM EDTA, 1 mM DTT and 0.3 mM PMSF) and clarified (21,500 g , 20 min). The recombinant proteins were purified from the clarified lysate with glutathione Sepharose04B beads (GSH beads, GE Healthcare) according to the manufacturer's instructions. A HeLa cell lysate was prepared as reported previously (Hawryluk-Gara et al., 2005 (link)). Briefly, asynchronous or nocodazole-arrested HeLa cells were lysed with lysis buffer A [10 mM Tris-HCl, pH 7.4, 400 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM DTT, complete proteinase inhibitor cocktail (Roche) and PhosSTOP (Roche)], sonicated, incubated for 30 min at 4°C and clarified (21,500 g , 20 min at 4°C). The clarified supernatant was diluted 3.75-fold with dilution buffer (10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 1 mM DTT, complete proteinase inhibitor cocktail and PhosSTOP), added to GSH beads that had been conjugated with GST proteins and incubated for 1 h at 4°C. The bound proteins were analyzed by western blotting.
8
GST-Trx Fusion Protein Purification
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Escherichia coli strain DH5α, transformed with pGEX6P-Trx (WT or its mutant derivatives), was precultured in 25 ml of LB/Ampicillin (100 μg ml−1) at 37°C for overnight. Each cultured medium was transferred to a fresh LB/ampicillin medium. After 37°C incubation for 3 hours, each cultured medium was treated with isopropyl-β-d (−)-thiogalactopyranoside (0.1 mM) and further incubated at 30°C for 2 hours. DH5α cells were harvested by centrifugation and resuspended in GST buffer. Cells were sonicated and treated with TX-100 (final concentration, 1%), and the cell lysates were incubated with Glutathione Sepharose 4B (GSH) beads (GE Healthcare) at 4°C for overnight. Recombinant protein–conjugated GSH beads were washed once with ice-cold tris-buffered saline, 0.1% tween 20 (TBST) and twice with ice-cold tris-buffered saline. To remove GST-tag from GST-Trx to obtain tag-free Trx, 16 U of PreScission protease (GE Healthcare) was added to GST-Trx–conjugated GSH beads in PreScssion buffer. After overnight incubation at 4°C, the supernatant was incubated with the reducing reagent DTT (2 mM) for 30 min on ice and dialyzed with Micro Float-A-Lyzer (Spectrum) at 4°C for overnight. Dialyzed liquid was then concentrated by Vivaspin 500 (Sartorius). The purified, reduced form of Trx was used for the experiments.
9
Protein Binding Affinity Assay
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The proteins were added to transport buffer (20 mM HEPES/NaOH, pH 7.4, 110 mM potassium acetate, 2 mM magnesium acetate, 5 mM sodium acetate, 0.5 mM EGTA/NaOH, 2 mM DTT) in the presence or absence of anti-importin α1 mAb, and mixed with glutathione-sepharose 4B beads (GSH-beads, GE Healthcare) or heparin-sepharose beads (Sigma). The mixtures were incubated at 4 °C for 1 h and then washed with transport buffer. Bound proteins were eluted with sample buffer for sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
10
Nucleotide Loading and Protein Binding Assay for GST-Rap1
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GST-Rap1 was loaded with GppNHp or GDP by incubating it in the presence of 10 mM EDTA and a 20-fold excess of the nucleotide for 2 h at room temperature [11 (link)]. Nucleotide binding to the G-proteins was stabilized by addition of MgCl2 to a final concentration of 20 mM. The G-proteins were prebound to GSH beads (GE Healthcare) by incubation for 2 h at 4 °C. The G-protein-coupled beads were washed with assay Buffer (50 mM Tris pH 7,5, 50 mM NaCl, 5 mM DTT, 10 mM MgCl2) and subsequently incubated with 50 μg of RA-TalB protein for 2 h at 4 °C. The GSH beads were harvested by centrifugation and washed three times in assay buffer. The samples were boiled in 1xSDS loading buffer and subjected to SDS-page.
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