Gfp trap resin

Manufactured by Proteintech

Sourced in Germany

The GFP-Trap resin is a laboratory tool designed for the affinity purification of Green Fluorescent Protein (GFP) and GFP-fusion proteins from cell lysates or other biological samples. The resin is pre-coated with specific antibodies that recognize and bind to the GFP tag, allowing the targeted protein to be isolated and concentrated for further analysis or applications.

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Lab products found in correlation

Phosphatase inhibitor, by Roche (2 mentions) Complete without edta, by Roche (2 mentions) Anti ha affinity matrix, by Roche (2 mentions) Mini protean tgx precast polyacrylamide gels and nitrocellulose membranes, by Bio-Rad (2 mentions) Versadoc imaging station, by Bio-Rad (2 mentions) Protease and phosphatase inhibitor cocktail, by Roche (2 mentions) Gfp trap a lysis buffer, by Proteintech (2 mentions) Gfp trap a wash buffer, by Proteintech (2 mentions) Anti flag monoclonal antibody, by Merck Group (2 mentions) Hek293t cell, by American Type Culture Collection (1 mentions) Profection mammalian transfection system, by Promega (1 mentions) Varian cary eclipse spectrofluorometer, by Agilent Technologies (1 mentions) Bca method, by G Biosciences (1 mentions) Flag m2 magnetic beads, by Merck Group (1 mentions) Gtpγs, by Merck Group (1 mentions) Anti gfp polyclonal antibody, by Merck Group (1 mentions) Anti lhcb2, by Agrisera (1 mentions) Anti myc, by Thermo Fisher Scientific (1 mentions) Protein a agarose, by Merck Group (1 mentions) Anti ha antibody, by Roche (1 mentions)

28 protocols using gfp trap resin

GFP-Trap Affinity Purification of Tepsin and LC3B

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For transient transfections, wild-type HeLa cells were seeded on six well plates and used the following day. pEGFP-N1 was transfected using Fugene 4K (Promega) at 1.5:1 Fugene:DNA ratio following manufacturer protocol. Cells were allowed to incubate for 24 h before use in immunoprecipitation assays.
Cell lysates from tepsin-GFP, pEGFP-N1 transiently-transfected, or mRFP-GFP-LC3B HeLa cells were prepared as described above. Lysate was incubated with unconjugated agarose (control) resin (Chromotek) for 30 min at 4°C to reduce background binding. Tepsin-GFP lysate and pEGFP-N1 transfected precleared lysate added to GFP-trap resin (Chromotek) at a ratio normalized by GFP transfection efficiency. For mRFP-GFP-LC3B, precleared lysate was divided equally among GFP-trap resin (Chromotek) and fresh control resin for coimmunoprecipitation (IP) experiments. Dilution buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 1 cOmplete Mini EDTA-free Protease Inhibitor Cocktail tablet per 20 ml) was added to bring each sample to an equal volume, then IPs were incubated 1 h at 4°C followed by three washes with dilution buffer. IPs were eluted by adding SDS loading buffer to the washed resin pellet and boiling for 5 min at 95°C.

Wallace N.S., Gadbery J.E., Cohen C.I., Kendall A.K, & Jackson L.P. (2024). Tepsin binds LC3B to promote ATG9A trafficking and delivery. Molecular Biology of the Cell, 35(4), ar56.

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Affinity Purification of GFP Fusion Proteins

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To purify GFP or GFP fusion protein complexes from seedlings, seven-day-old seedlings were harvested and ground in liquid N₂ and then incubated at 4°C for 1h with lysis buffer 25 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.5% NP40, containing protease inhibitor complex (Roche). Co-immunoprecipitation was performed using 25μl GFP-trap resin (ChromoTek) and incubated with cell lysate at 4°C for 1h. The beads were recovered by centrifugation at 1,000xg for 2 min and washed 4 times with 500μl wash buffer 25 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.1% NP40. Bound proteins were released by incubation with 0.1 M glycine, pH2.5 or analyzed using 2xSDS buffer. Eluents were analyzed by SDS-PAGE and immunoblotting or by LC-MS/MS analysis (SPARC BioCentre, Sick Kids, Toronto).

Jiang T., Oh E.S., Bonea D, & Zhao R. (2017). HSP90C interacts with PsbO1 and facilitates its thylakoid distribution from chloroplast stroma in Arabidopsis. PLoS ONE, 12(12), e0190168.

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In Vitro Ubiquitination of GFP-PTOV1

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In vitro ubiquitination assays were performed as previously described (40 (link), 41 (link)). In brief, recombinant human HUWE1 HECT domain was produced and purified from BL21 bacterial cells. Recombinant E1, UbcH7 (E2), and ubiquitin proteins were purchased from R&D Systems. GFP-PTOV1 was overexpression in HEK-293T cells by transiently transfecting 16 ug of pcDNA3 encoding GFP-PTOV1 into 1×10⁶ cells in a 10cm dish. 48 hours later, cells were lysed and GFP-PTOV1 was retrieved with GFP-TRAP resin (Chromotek, Planegg, Germany). After washing, GFP-PTOV1 on beads was incubated at 30°C for 3 hours with 10 ng of recombinant E1, 100 ng of recombinant UbcH7, 100 μg of ubiquitin, and 1 μg of a purified HECT domain of HUWE1 in 40 μl of reaction buffer [50 mM tris (pH 7.5), 5 mM MgCl2, 2 mM ATP, 2 mM DTT]. After the incubation, the beads were washed 3X with a buffer containing 10 mM Hepes (pH 7.4), 150 mM KCl, 1% NP-40, and 400 mM NaCl. GFP-PTOV1 protein was eluted with Laemmli SDS sample buffer and subjected to Western blot.

Pennington K.L., McEwan C.M., Woods J., Muir C., Sahankumari A.G., Eastmond R., Balasooriya E.R., Egbert C.M., Kaur S., Heaton T., McCormack K.K., Piccolo S.R., Kurokawa M, & Andersen J.L. (2021). SGK2, 14-3-3 and HUWE1 cooperate to control the localization, stability and function of the oncoprotein PTOV1. Molecular cancer research : MCR, 20(2), 231-243.

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Protein Extraction and Affinity Purification

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Total yeast protein extracts were prepared using the same protocols described earlier. A 10-μl amount of GFP-Trap resin (ChromoTek) was used for 1 mg of each cell lysate and agitated at 4°C for 2 h. Bound protein was eluted with 50 μl of hot (95°C) SDS–PAGE loading buffer. Eluted proteins were resolved on 7–15% split SDS gels.

Lwin K.M., Li D, & Bretscher A. (2016). Kinesin-related Smy1 enhances the Rab-dependent association of myosin-V with secretory cargo. Molecular Biology of the Cell, 27(15), 2450-2462.

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GFP-SMEK1 Protein Interaction Study

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The HEK293T cells (ATCC, tested to be void of mycoplasma contamination) were grown in advanced Dulbecco’s modified Eagle’s medium/F12 supplemented with 10% fetal bovine serum and then transfected in 9 cm dishes with 15 μg of plasmid DNA for GFP-SMEK1 (Addgene), using the Profection mammalian transfection system (Promega). 12 h post transfection, medium was replaced with fresh antibiotic-free media and LY294002 was added at a final concentration of 20 μM. After 48 h of LY294002 treatment, cells were harvested by scraping and frozen in liquid nitrogen. For the co-IPs, these cell pellets were thawed into lysis buffer (50 mM HEPES at pH 7.4, 1 mM EGTA, 1 mM MgCl2, 150 mM KCl, 10% (v/v) glycerol, Complete (Roche), 1 mM phenylmethyl sulphonyl fluoride, phosphatase inhibitors (Roche), and 0.5% (v/v) NP-40) and lysed by shearing through a G30 syringe. Lysates were cleared at 20,000 g. GFP-tagged proteins were immunoprecipitated using GFP-Trap resin (Chromotek) and eluted by boiling in 2× sample buffer. Input (IN) and eluate (IP) samples were analyzed by SDS-PAGE and western blotting.

Sen I., Zhou X., Chernobrovkin A., Puerta-Cavanzo N., Kanno T., Salignon J., Stoehr A., Lin X.X., Baskaner B., Brandenburg S., Björkegren C., Zubarev R.A, & Riedel C.G. (2020). DAF-16/FOXO requires Protein Phosphatase 4 to initiate transcription of stress resistance and longevity promoting genes. Nature Communications, 11, 138.

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S1P Receptor Immunoprecipitation and Analysis

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Cells expressing S1PR1-eGFP or mS1PR1-eGFP were S1P-starved and mock-stimulated or stimulated with the indicated agonists for 5 or 30 min; 10⁶ cells were used for each experimental point. Cells were washed twice in ice-cold phosphate-buffered saline and lysed in 20 mM HEPES, 150 mM NaCl, 1% Triton X-100, 0.15% Nonidet P-40, 1× HALT™ Proteases and Phosphatases inhibitor cocktail, 1.25 U/mL Benzonase. Clarified lysates were incubated for 1 h at +4 °C with GFP-Trap® resin (Chromotek) and extensively washed with lysis buffer. Finally, the immunopurified material was eluted in 4% SDS and analysed by immunoblot. The equal loading was based on the GFP amount reading the samples in the Cary Eclipse Varian spectrofluorometer with 488 nm λ_Ex and 509 nm λ_Em. In some experiments, cells expressing S1PR1 without the GFP tag were used as control and the equal loading was based on total protein concentration measured with the BCA method (G Biosciences).

Patrone M., Cammarota E., Berno V., Tornaghi P., Mazza D, & Degano M. (2020). Combinatorial allosteric modulation of agonist response in a self-interacting G-protein coupled receptor. Communications Biology, 3, 27.

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Immunoprecipitation of FLAG- and GFP-tagged proteins

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At 48 h after plasmid transfection in 10 cm dish, HEK293T cells were washed twice with ice-cold phosphate-buffered saline. Cells were scraped from dish using 1 ml cold PBS and then were spun at 500 g for 3 min at 4°C. After removal of the supernatant, the cell pellet were lysed in 600 μl IP lysis buffer (250 mM NaCl, 50 mM pH 7.5 Tris–HCl, 1 mM EDTA, 10% glycerol, 0.5% NP40) containing EDTA-free protease inhibitor (Roche) at 4°C for 30 min. Centrifuge cell lysate at 20 000 g for 10 min at 4°C and transfer supernatant to a pre-cooled tube. The FLAG^® M2 magnetic beads (Sigma-Aldrich) or GFP-Trap resin (ChromoTek) were washed three times using the IP lysis buffer and 25 μl beads were added to each supernatant tube. After incubating for 4 h at 4°C, the beads were washed three times with the IP lysis buffer and then were boiled with 100 μl 2× SDS-sample buffer for 10 min at 95°C to dissociate immunocomplexes.

Li N., Wang J., Wallace S.S., Chen J., Zhou J, & D’Andrea A.D. (2020). Cooperation of the NEIL3 and Fanconi anemia/BRCA pathways in interstrand crosslink repair. Nucleic Acids Research, 48(6), 3014-3028.

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NEIL3-EGFP Interactor Identification

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HEK293T cells were seeded in 15 cm dishes and transfected with NEIL3-EGFP or empty vector. Twenty four hours after transfection, cells were treated with 10 μM psoralen plus UVA irradiation (0.8 J/cm²) and released to culture for indicated time. Cells were then washed with pre-chilled PBS three times and lysed into the lysis buffer (20 mM HEPES-K⁺, pH 7.6, 0.1 mM KCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 10% glycerol, 0.01% NP-40, 1 mM DTT, 0.2 mM PMSF, 0.5 mM benzamidine, 1 ug/ml each of leupeptin, aprotinin and pepstatin A) for 30 min at 4°C. Cell lysates were incubated with GFP-Trap resin (ChromoTek) at 4°C overnight and the resin was washed with cold lysis buffer four times. The immunoprecipitated NEIL3-eGFP and its binding partners were eluted with SDS elution buffer (2% SDS, 50 mM Tris–Cl, pH 6.8). The samples were prepared by trichloroacetic acid (TCA) precipitation as previously described (42 (link)), and sent to for mass spec analysis.

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Co-immunoprecipitation of CBX4, SALL1 and Variants

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HEK 293FT cells were plated at 25–30% confluence. Transient transfections were performed using calcium phosphate in a 10 cm dish with 5 μg of CMV-CBX4-YFP, CMV-SALL1-YFP, CMV-SALL1ΔSUMO-YFP, CMV-SALL1ΔSIM-YFP, CMV-YFP, CMV-SALL1-2xHA, CMV-SALL1-826-2xHA, CB6-HA, CB6-HA-SALL1, CB6-HA-SALL1ΔSUMO or CB6-HA-CBX4 in complete medium. All steps after transfection were performed at 4°C. Two days after transfection, cells were washed three times with cold 1× PBS and detached from the dish with a scraper. Cells of 10 cm dishes were lysed by adding 1 ml of Lysis Buffer [25 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% Triton X-100, 5% glycerol, protease inhibitors (Roche)] followed by incubation on a rotating wheel for 30 min at 4°C. Lysates were sonicated and spun down at 25,000 × g for 20 min. After saving 40 μl of supernatant (input), the rest of the lysate was incubated overnight with 30 μl of equilibrated GFP-Trap resin (Chromotek) in a rotating wheel. Beads were washed five times for 5 min each with washing buffer (25 mM Tris–HCl pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% Triton X-100, 5% glycerol). Beads were centrifuged at 2000 × g for 2 min after each wash. For elution, samples were boiled for 5 min at 95°C in 2× Laemmli buffer.

Giordano I., Pirone L., Muratore V., Landaluze E., Pérez C., Lang V., Garde-Lapido E., Gonzalez-Lopez M., Barroso-Gomila O., Vertegaal A.C., Aransay A.M., Rodriguez J.A., Rodriguez M.S., Sutherland J.D, & Barrio R. (2021). SALL1 Modulates CBX4 Stability, Nuclear Bodies, and Regulation of Target Genes. Frontiers in Cell and Developmental Biology, 9, 715868.

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GFP Fusion Protein Interactome Analysis

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HeLa cells expressing GFP fusion proteins together with dsRed2-IPO13 treated with and without H₂O₂ were lysed 12 h post-transfection. In some samples, lysates were preincubated with GTPγS at a final concentration of 1.7 mM (Sigma-Aldrich) for 20 min on ice. Immunocomplexes were precipitated and eluted using GFP-Trap resin (ChromoTek) according to manufacturer instructions. Input and immunoprecipitations were analysed by Western blotting.

Gajewska K.A., Lescesen H., Ramialison M., Wagstaff K.M, & Jans D.A. (2021). Nuclear transporter Importin-13 plays a key role in the oxidative stress transcriptional response. Nature Communications, 12, 5904.

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