DY47304 was grown to mid‐log phase in EMM medium at 30 °C. About 2000 OD600 units of cells were harvested and washed once with water. Cells were lysed by grinding in liquid nitrogen. The resulting powder was mixed with lysis buffer (50 mm HEPES‐NaOH, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 0.05% NP‐40, 10% glycerol, 1× Roche protease inhibitor cocktail). After centrifugal clarification, the cell lysate was incubated with GFP‐Trap Sepharose beads (ChromoTek, Planegg‐Martinsried, Germany) for 3 h at 4 °C. The beads were washed 4 times with wash buffer (50 mm HEPES‐NaOH, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 0.05% NP‐40, 10% glycerol) and incubated with 100 μL of lysis buffer and 2 μg of 3C protease overnight at 4 °C. Protease‐released proteins were concentrated and buffer‐exchanged to storage buffer (50 mm Tris/HCl, pH 7.5, 150 mm NaCl, 5 mm MgCl2) by using an Amicon Ultra‐0.5 centrifugal filter with 30 kDa molecular weight cutoff (Millipore, Burlington, MA, USA).
Gfp trap sepharose beads
Manufactured by Proteintech
Sourced in Germany
GFP-Trap Sepharose beads are a laboratory tool used for the purification and detection of green fluorescent protein (GFP) and GFP-fusion proteins. The beads are coated with a highly specific single-domain antibody that binds to GFP with high affinity, allowing for efficient capture and isolation of GFP-tagged proteins from complex biological samples.
Automatically generated - may contain errors
4 protocols using gfp trap sepharose beads
1
Purification of GFP-tagged Proteins
2
GFP-Trap Affinity Purification
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The equivalent of 100 OD600 growing cells was transferred to SD-N medium for 1 h, harvested by centrifugation and resuspended in 1 ml of lysis buffer (45 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Tween-20) supplemented with 1 mM PMSF, Complete protease inhibitors (Roche), 10 mM β-glycerophosphate, 50 mM NaF, and 1 mM Na3VO4. After addition of glass beads, cells were lysed by vortexing at 4 °C for 15 min and lysates cleared by centrifugation at 15 000 g for 5 min at 4 °C. The supernatant was incubated with 25 µl of pre-washed GFP-trap sepharose beads (ChromoTek) on a rotating wheel for 1.5 h at 4 °C. Beads were finally washed 3 times in 1 ml of lysis buffer, resuspended in loading buffer and analyzed by SDS-PAGE followed by western blot.
3
Purification of FW-MBP-GFP Fusion Protein
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Fission yeast cells expressing the CtAms1-CtNbr1 FW-MBP-GFP fusion protein from the Pnmt1 promoter were grown to mid-log phase in EMM medium at 30 °C. About 2000 OD600 units of cells were harvested and washed once with water. Cells were lysed by grinding in liquid nitrogen. The resulting powder was mixed with lysis buffer (50 mM HEPES-Na, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.05% NP-40, 10% glycerol, 1× Roche protease inhibitor cocktail). After centrifugal clarification, the cell lysate was incubated with GFP-Trap Sepharose beads (ChromoTek) for 3 h at 4 °C. The beads were washed 4 times with wash buffer (50 mM HEPES-Na, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.05% NP-40, 10% glycerol) and incubated with 100 μl of lysis buffer and 2 μg of 3 C protease at 4 °C overnight. The released CtAms1-CtNbr1 FW-MBP fusion protein was concentrated and buffer-exchanged to storage buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2) by using an Amicon Ultra-0.5 centrifugal filter with 30 kDa molecular weight cutoff (Millipore).
4
Co-Immunoprecipitation of Plant Proteins
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Co-IP was carried out as previously described (Heard et al., 2015) with minor modifications. Seven-day-old seedlings were ground in a prechilled mortar and pestle on ice with extraction buffer (150 mM Na-HEPES, pH 7.5, 10 mM EDTA, 10 mM EGTA, 17.5% [w/v] sucrose, 7.5 mM KCl, 0.01% [v/v] Igepal CA-630, 10 mM dithiothreitol, 1% [v/v] protease inhibitors [Sigma, St. Louis, MO, USA; Cat # 11836170001], 0.5% [v/v] polyvinylpolypyrrolidone), with 2 mL buffer per 1 g of tissue (fresh weight). The homogenate was filtered through two layers of Miracloth (Millipore, Burlington, MA, USA; Cat # 475855-1R), and the flow-through was centrifuged at 4°C, 6,000 g for 15 min. For the immunoprecipitation assay, 20 µL GFP-Trap Sepharose beads (ChromoTek, Munich, Germany; Cat # gta-20) were added to the supernatant, followed by incubation for 6 h at 4°C. The slurry was washed five times with prechilled extraction buffer (no polyvinylpolypyrrolidone or protease inhibitors). The slurry was collected after the last wash and proteins eluted with 5× SDS-PAGE loading buffer for immunoblot analysis or liquid chromatography-tandem MS (LC-MS/MS). The LC-MS/MS assay was conducted by Protein World Biotech Ltd. (Beijing, China).
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