Gst spin purification kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The GST Spin Purification Kit is a laboratory tool designed for the rapid purification of GST-tagged recombinant proteins. The kit utilizes glutathione-coated resin in a spin column format to efficiently capture and elute the target protein. This product provides a convenient and reliable method for the isolation of GST-fusion proteins from cell lysates or other complex samples.

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Lab products found in correlation

Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Gst protein interaction pull down kit, by Thermo Fisher Scientific (1 mentions) Dmem without phenol red, by Thermo Fisher Scientific (1 mentions) 20 3h phorbol 12 13 dibutyrate 3h pdbu, by PerkinElmer (1 mentions) Phorbol 12 myristate 13 acetate, by LC Laboratories (1 mentions) Qiaquick pcr purification kit, by Qiagen (1 mentions) Hek293 cell, by American Type Culture Collection (1 mentions) Qiaprep spin miniprep kit, by Qiagen (1 mentions) Rpmi 1640 medium, by American Type Culture Collection (1 mentions) Fetal bovine serum (fbs), by American Type Culture Collection (1 mentions) L glutamine, by American Type Culture Collection (1 mentions) Lncap human prostate cancer cells, by American Type Culture Collection (1 mentions) Neuro2a mouse neuroblastoma cells, by American Type Culture Collection (1 mentions) B per bacterial protein extraction reagent, by Thermo Fisher Scientific (1 mentions) One shot bl21 de3 chemically competent e coli, by Thermo Fisher Scientific (1 mentions) Isopropyl o d thiogalactopyranoside, by Thermo Fisher Scientific (1 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by GoldBio (1 mentions) Amicon centrifugal filter unit, by Merck Group (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions) Ripa buffer, by Beyotime (1 mentions)

8 protocols using gst spin purification kit

GST-NEDD4L Protein Purification and Pull-Down

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The expression vectors with either GST-labeled NEDD4L or His-labeled SP1 were constructed by Shanghai Genechem Co., Ltd. GST Spin Purification Kit (16107) and GST Protein Interaction Pull-Down Kit (21516) were purchased from Thermo.
Protein purification: briefly, centrifuging column at 700 × g for 2 min to remove storage buffer and equilibrate column with two resin-bed volumes of equilibration buffer. Add the prepared protein extract to the column and allow it to enter the resin bed. Then centrifuging column at 700 × g for 2 min and collect the flow-through in a centrifuge tube. Washing resin with two resin-bed volumes of equilibration buffer. Centrifuging at 700 × g for 2 min and collect fraction in a centrifuge tube. Eluting GST-tagged protein from the resin by adding one resin-bed volume of elution buffer. Protein pull-down assay: briefly, immobilizing the obtained GST-NEDD4L protein on the glutathione agarose according to the instructions. Adding in prepared prey protein sample and incubated at 4 °C for at least 1 h. Centrifuging at 1250 × g for 30 s to 1 min. Adding in 400µL of wash solution and repeat washing for a total of five washes. Eluting with glutathione elution buffer and collecting eluent for analysis.

Cui J., Shu C., Xu J., Chen D., Li J., Ding K., Chen M., Li A., He J., Shu Y., Yang L., Zhang R, & Zhou J. (2020). JP1 suppresses proliferation and metastasis of melanoma through MEK1/2 mediated NEDD4L-SP1-Integrin αvβ3 signaling. Theranostics, 10(18), 8036-8050.

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Protein Interaction Detection via Pull-Down Assay

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The coding regions of MdSnRK1.1 and MdJAZ18 were introduced into the PET-32a and PGEX-4T-1 vector, respectively, and then recombinant vector was transformed into Escherichia coli BL21(DE3) to express HIS-MdSnRK1.1 or GST–MdJAZ18 protein. The pull-down assay was performed according to the instructions of the Pierce GST Spin Purification Kit (Thermo, MA, USA). Then samples were detected by immunoblotting with anti-GST and anti-HIS antibodies, respectively.

Liu X.J., An X.H., Liu X., Hu D.G., Wang X.F., You C.X, & Hao Y.J. (2017). MdSnRK1.1 interacts with MdJAZ18 to regulate sucrose-induced anthocyanin and proanthocyanidin accumulation in apple. Journal of Experimental Botany, 68(11), 2977-2990.

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Purification and Expression of ZIKV RNA Polymerase

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ZIKV RNA polymerase (ZVRP) was obtained from ZIKV-infected BHK-21 cells. The cells were infected with ZIKV at an MOI of 10 for 24 h, lysed with buffer A [containing 0.25 M potassium phosphate (pH 7.5), 10 mM 2-mercaptoethanol (2-ME), 1 mM EDTA, 0.5% Triton X-100, 0.5 mM phenylmethane sulfonyl fluoride (PMSF) and 20% glycerol], sonicated and centrifuged at 10,000 × g for 10 min at 4 °C. The resulting supernatant was further centrifuged at 100,000 × g for 90 min at 4 °C and passed through two ion-exchange columns, DEAE- and phospho-cellulose19 (link). Alternatively, the ZIKV NS5 region encoding the nucleotides responsible for the RNA-dependent RNA polymerase (RDRP) activity were cloned into the pET-41b+ vector (Novagen) between the BamHI and SacI sites. ZVRP expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to the E. coli strain BL21. The cells were lysed in buffer A, and the N-terminal GST-tag was used to purify the protein using a GST spin purification kit (ThermoFisher Scientific) according to the manufacturer’s instructions.

Sacramento C.Q., de Melo G.R., de Freitas C.S., Rocha N., Hoelz L.V., Miranda M., Fintelman-Rodrigues N., Marttorelli A., Ferreira A.C., Barbosa-Lima G., Abrantes J.L., Vieira Y.R., Bastos M.M., de Mello Volotão E., Nunes E.P., Tschoeke D.A., Leomil L., Loiola E.C., Trindade P., Rehen S.K., Bozza F.A., Bozza P.T., Boechat N., Thompson F.L., de Filippis A.M., Brüning K, & Souza T.M. (2017). The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication. Scientific Reports, 7, 40920.

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Purification and Labeling of Proteins

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[20-³H]Phorbol 12,13-dibutyrate ([³H]PDBu) (17.2 Ci/mmol) was obtained from PerkinElmer Life Sciences. PDBu and phorbol 12-myristate 13-acetate (PMA) were purchased from LC Laboratories (Woburn, MA). Phosphatidyl-L-serine (PS), phosphatidylcholine (PC), and sphingosine were from Avanti Polar Lipids (Alabaster, AL) and the isopropyl O-D-thiogalactopyranoside (IPTG) was from Sigma (St. Louis, MO). LNCaP human prostate cancer cells, Neuro-2a mouse neuroblastoma cells, HEK-293 cells, fetal bovine serum (FBS), RPMI 1640 medium, and L-glutamine were from the American Type Culture Collection (Manassas, VA). Reagents used for culturing bacteria (LB Broth, LB agar plates with ampicillin, ampicillin solution, etc.) were from K-D Medical, Inc. (Columbia, MD). The primers, the High Fidelity Polymerase, and the ligase used for cloning, the chemically competent MaxEfficiency DH5αT1^R and BL-21(DE3) cells, the DMEM without phenol red used for confocal analysis, the Lipofactamine and Plus reagent, Lipofectamine 3000, and the GST spin purification kit were from Thermo Scientific (Pittsburgh, PA). The QIAquick PCR Purification Kit and the QIAprep Spin Miniprep Kit were from Qiagen (Germantown, MD). The pHTN HaloTag(R) CMV-neo vector and the kit for purification of Halo-tagged proteins were from Promega (Madison, WI).

Das J., Kedei N., Kelsey J.S., You Y., Pany S., Mitchell G.A., Lewin N.E, & Blumberg P.M. (2018). Critical Role of Trp-588 of Presynaptic Munc13-1 for Ligand Binding and Membrane Translocation. Biochemistry, 57(5), 732-741.

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Purification of RasGRP1/3 C1 and REM Domains

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The C1 domain and REM motif of RasGRP1/3 in the pGEX-2T and pGEX-5X1 plasmids were transformed into BL21 (DE3) One Shot chemically competent E. coli (Invitrogen). Transformants were grown in LB broth medium (K-D Medical) at 37°C until the optical density of the bacterial suspension reached 0.6–0.8. Expression of the GST fusion proteins was induced with 0.3 mM isopropyl O-D-thiogalactopyranoside (Thermo Fisher Scientific) for 4 h at 37°C or 6 h at room temperature (C1 domains and REM motifs, respectively). Bacterial cells were subjected to B-PER bacterial protein extraction reagent (Thermo Fisher Scientific) or lysis buffer (150 mM NaCl, 50 mM Tris buffer, pH 7.4). The expressed GST-tagged C1 and REM proteins were purified using a GST Spin Purification Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Purification efficiency was evaluated by SDS-PAGE analysis. Purified proteins were stored in 20% glycerol at −80°C.

Czikora A., Kedei N., Kalish H, & Blumberg P.M. (2017). Importance of the REM (Ras exchange) domain for membrane interactions by RasGRP3. Biochimica et biophysica acta, 1859(12), 2350-2360.

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Recombinant Protein Purification Protocol

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All plasmids were shuttled into the bacterial expression vector pEX-N-His-GST (PS100028, Origene) using T4 DNA Ligase (New England Biolabs) and AsiSI and MluI-HF restriction enzymes (New England Biolabs) before protein expression and purification. An overnight culture of transfected E. coli cells was added to 250 mL LB broth at a 1:100 dilution and then incubated for 3 h at 37 °C with shaking. Cells were induced with 1 mM IPTG (GoldBio, Olivette, MO, USA) for 2 h at 37 °C with shaking. Induced LB broth was centrifuged, and the resulting pellet was resuspended in B-PER (Pierce) and allowed to incubate at room temperature with shaking for 15 min. This mixture was centrifuged, and the resulting cell lysate was then used in the GST Spin Purification kit (#16108, Thermo Fisher), following the manufacturer’s instructions, to purify recombinant protein. Purified protein was concentrated using Amicon™ Centrifugal Filter units (MilliporeSigma™, Burlington, MA, USA), aliquoted and spiked with 10% glycerol, and flash frozen into stocks that were kept at −80 °C until use.

Nauman M.C., Won J.H., Petiwala S.M., Vemu B., Lee H., Sverdlov M, & Johnson J.J. (2023). α-Mangostin Promotes In Vitro and In Vivo Degradation of Androgen Receptor and AR-V7 Splice Variant in Prostate Cancer Cells. Cancers, 15(7), 2118.

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GST-HIP1R Protein Purification and Binding

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Glutathione S-transferase (GST)-HIP1R_1–632, GST-HIP1R_633–822 and GST-HIP1R_823–1068 motif were expressed in E. coli strain BL21 (DE3) and induced using 0.1 mM IPTG at 16°C overnight. Proteins were purified using GST Spin Purification kit (16106, Thermo Fisher Scientific, Waltham, MA, USA) in the presence of protease inhibitor cocktail tablets (04693116001, Roche Diagnostics, Indianapolis, IN, USA) according to manufacturer’s protocols. High density cultured neurons were lysed with RIPA buffer (Beyotime, Shanghai, China) and incubated with 2 μg of purified proteins overnight at 4°C. Forty microliter glutathione sepharose beads were added and the mixture was incubated for another 3 h at 4°C. After washing for five times, beads were resuspended in 40 μl sample buffer for western blot analysis.

Yang Q., Peng L., Wu Y., Li Y., Wang L., Luo J.H, & Xu J. (2018). Endocytic Adaptor Protein HIP1R Controls Intracellular Trafficking of Epidermal Growth Factor Receptor in Neuronal Dendritic Development. Frontiers in Molecular Neuroscience, 11, 447.

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Recombinant Protein Pulldown Assay

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The pGEX‐4‐T‐1‐TMEM182, pGEX‐4‐T‐1‐ITGB1, or empty pGEX‐4‐T‐1 were transformed into Escherichia coli BL21DE3 pLys (Thermo, San Jose, CA, USA). Bacteria were grown to an OD600 of 0.8 and then induced with 0.5 mM of IPTG (Sigma) for 2 h at 37°C in a shaking incubator. TMEM182‐GST protein and ITGB1‐GST protein were isolated using a GST spin purification kit (Thermo). TMEM182‐GST and ITGB1‐GST were incubated with total proteins extracted from indicated treated chicken myoblasts or control chicken myoblasts and rotated overnight at 4°C in binding/washing buffer [50 mm Tris (pH 7.5), 0.1 mm Ethylenediaminetetraacetic acid, 1% Triton X‐100, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, and 1 mm DTT]. To pull down GST, Glutathione agarose beads (Thermo) were added the next day and allowed to incubate for 2 h at 4°C and then washed with the washing buffer. Samples were eluted by incubation with Laemmli sample buffer (Bio‐rad, CA, USA) and boiling for 5 min. Samples were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Immunoblotting was performed against FLAG (1:5000, A02010, Abbkine, Guangzhou, China) to detect FLAG‐tagged protein and against GST to detect GST protein as a loading control.

Luo W., Lin Z., Chen J., Chen G., Zhang S., Liu M., Li H., He D., Liang S., Luo Q., Zhang D., Nie Q, & Zhang X. (2021). TMEM182 interacts with integrin beta 1 and regulates myoblast differentiation and muscle regeneration. Journal of Cachexia, Sarcopenia and Muscle, 12(6), 1704-1723.

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