TNF‐α, PeproTech (300‐01A); HALO‐link resin, Promega (G1913); MLN7243, Active Biochemicals (A‐1384); MLN4924, Active Biochemicals (A‐1139); H2O2, Sigma (216763); Camptothecin, Sigma (C9911); Cycloheximide, Sigma (C1988); DPQ, Calbiochem (300270); CellTiter‐Glo, Promega (G7570); Olaparib, Cambridge Biosciences (CAY10621); PARG inhibitor PDD 17273, Tocris (5952); Sodium Butyrate, Sigma (303410); GFP‐Trap_A, Chromotek (gta), PARP‐1‐Trap_A, Chromotek (xta); phosSTOP, Sigma (4906845001); cOmplete EDTA‐free protease inhibitor cocktail (Roche‐11836170001); Pepstatin A, Sigma (P5318), Bestatin hydrochloride, Sigma (B8385); Deoxyribonuclease I, Sigma (D5025); LDS Sample Buffer, Life Technologies (NP0007); Colloidal Coomassie, Expedeon (ISB1L); IPTG, Formedium (IPTG025); Micrococcal Nuclease, NEB (M0247S).
Gfp trap a
Manufactured by Proteintech
Sourced in Germany
GFP-Trap_A is a laboratory equipment designed for the efficient purification of GFP-fusion proteins from cell lysates or other biological samples. It utilizes the high-affinity interaction between the GFP-tag and the GFP-binding protein to capture and isolate the target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (5 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Complete protease inhibitor cocktail, by Roche (4 mentions)
Xtremgene hp reagent, by Merck Group (3 mentions)
Horseradish peroxidase hrp conjugated donkey anti rabbit igg, by GE Healthcare (2 mentions)
Anti gfp, by Santa Cruz Biotechnology (2 mentions)
Ezview red anti ha affinity gel, by Merck Group (2 mentions)
Anti flag m2 magnetic bead, by Merck Group (2 mentions)
Proteinase phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (2 mentions)
Laemmli 2x concentrate, by Merck Group (2 mentions)
Rfp trap a, by Proteintech (2 mentions)
Bab agarose beads, by Proteintech (2 mentions)
Immobilon p pvdf transfer membrane, by Merck Group (2 mentions)
Benzonase, by Merck Group (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Anti flag m2 affinity gel, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Merck Group (2 mentions)
78 protocols using gfp trap a
1
Proteomic Analysis of Cellular Stress Response
2
Cross-Linking and Affinity Purification of RNA-Protein Complexes
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Strains expressing HisRS mRNA variants (WT, GAG, Stem disrupt, and Stem unchange) tagged at the 3' UTR with multiple MS2 loops and expressing pCP-GFP [58 (link)] were grown to logarithmic phase (OD600 0.8–1) in 500 ml. For RNA “Input” sample, 10 ml were set aside, and RNA were extracted by the hot phenol method [59 (link)]. The remainder of cells (490 ml) were subjected to cross-linking by addition of formaldehyde (0.05% final concentration) in PBS and slowly rotated at room temperature for 10 min to cross-link protein–RNA complexes. 0.125 M Glycine was added to stop the cross-linking reaction. Cells were then suspended in 1 ml cold buffer B (with 5 mg/ml Heparin final concentration). Cells were lysed and cleared as in RIP method. Aliquot (1/20 of the lysate) were set aside for proteins' “Input” sample. Lysates were loaded on GFP-Trap_A (ChromoTek) and rotated at 4°C for 2 hr. Samples were washed four times in wash buffer (20 mM Tris-HCl [pH 7.5], 0.5 M NaCl, 0.5% NP40, 0.1% SDS, 0.5 mM EDTA, 0.5 mM DTT, 0.01 U/μl RiboLock RNase Inhibitor [Thermo Scientific]). Proteins were eluted from 80% of the beads with LSB and resolved on 10% PAGE. RNA was extracted from 20% of the beads by 0.2 M Glycine buffer (pH 2.5). Cross-linking was reversed by heating at 65°C for 2 hr in reverse cross-linking buffer, and RNA was precipitated after phenol:choloroform extraction.
3
GFP-based Immunoprecipitation Protocol
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Cells cultured in 6-well plates were disrupted with 300 μl of cold lysis buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5% NP40) supplemented with a protease inhibitor mix (GE Healthcare). Lysates were preclarified by centrifugation (14,000 g, 4 °C, 15 min). Immunoprecipitations were performed using purified GFP Binding Protein (GBP)46 (link) bound covalently to AminoLink resin, according to the manufacturer’s instructions (Thermo Scientific Pierce), after pre-clearing with AminoLink resin alone. 200 μl of cell lysate was incubated with 25 μl of GBP-AminoLink resin for 1h30min at 4 °C under constant stirring. Alternatively, GFP-tagged proteins were immunoprecipitated with magnetic GFP-Trap®_A (Chromotek, Planegg, Germany). After extensive washing with cold W100 buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA), immunoprecipitates were finally eluted from the beads by boiling for 5 min in Laemmli sample buffer, then were submitted to Western blot analysis.
4
Regulation of HERC2 and p53 Interactions
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The following reagents were used: anti-HERC2 monoclonal (BD Biosciences #612366); anti-HERC2 polyclonal (Bvg1 antibodies against residues 4785-4834, and Bvg9 antibodies against residues 1-199) [15 (link)]; anti-p21 (C-19), anti-p53 (FL-393), anti-NEURL4 (E-20) (Santa Cruz Biotechnology, Inc.); anti-p53 Ab-5 (DO-7) (Neo Markers); anti-GST monoclonal (GenScript); anti-Ran [34 (link)]; anti-α-tubulin (Ab-1) (Calbiochem); anti-USP33 (Proteintech); anti-Flag M2 (Sigma); anti-GFP and anti-c-myc (clone 9E10) (Roche); anti-MDM2 (2A10) (Abcam); Z-Leu-Leu-Leu-al (MG132) (Sigma-Aldrich); horseradish peroxidase-conjugated secondary antibodies; lipofectamine LTX (Invitrogen); cycloheximide (Applichem); protein A-Sepharose and glutathione-Sepharose (GE Healthcare); GFP-Trap_A (ChromoTek); Immobilon-P PVDF transfer membrane (Millipore Corporation); luciferase assay system (Promega); luminescent β-galactosidase detection Kit II (Clontech Laboratories).
5
Nup188 Interacts with Centrosomal Proteins
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To test whether Nup188 biochemically interacted with Cep192, Cep152, and PCM1, we cotransfected HEK293T cells with pFLAG-NUP188 and pEGFP-C2, pGFP-Cep192 (pKM3105), pGFP-Cep152, or pGFP-PCM1. After 24 h, cells were washed with PBS and lysed in 50 mM Tris, pH 7.4, 250 mM NaCl, 1% Igepal, 1 mM PMSF, and protease and phosphatase inhibitors for 20 min on ice. The insoluble material was pelleted by centrifugation at 14,000 rpm for 10 min at 4° C, and the supernatant was incubated with anti-GFP nanobody-conjugated agarose beads (GFP-Trap_A; Chromotek) for 2 h at 4° C. Beads were then washed in lysis buffer three times and eluted with Laemmli sample buffer. Proteins in eluates were separated by SDS-PAGE gels and processed for Western blotting.
6
Characterizing Exportin Interactions
7
Immunoprecipitation of GFP-Interacting Proteins
8
Immunoprecipitation of GFP- and HA-tagged Proteins
9
Solubilization and Purification of hTRPV3
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Expression and isolation of crude membranes were performed as described above. For solubilization, 2% (w/v) DDM and 0.2% (w/v) CHAPS (anagrade, Affymetrix) were added to gently thawed membranes, and the sample was stirred for 1 h at 4 °C. The solubilizate was ultracentrifuged (60,000 × g for 1 h) to discard non-solubilized material and protein aggregates. The supernatant was incubated for 30 min with GPF-nanobody-coupled agarose beads (GFP-Trap_A, Chromotek) at 4 °C. Then the flow-through was discarded, and the beads were washed with 10 column volumes of buffer A, supplemented with 0.2% (w/v) DDM and 0.2% (w/v) CHAPS. To elute hTRPV3, the fusion protein was cleaved off overnight by incubation with PreScission protease (0.3 mg·ml−1) at 4 °C, leaving the GFP-His tag bound to the beads. The total elution (eluate + 5 column volumes of wash) was then concentrated to ∼1 ml (100 kDa cut-off, Vivaspin 20, Sartorius) and loaded on a Superose 6 10/300 GL gel filtration column (AKTA purifier system, GE Healthcare). The running buffer consisted of buffer B (100 mm NaCl, 10 mm Tris, pH 7.5), supplemented with 0.03% (w/v) DDM and 0.1% (w/v) CHAPS. After analysis on SDS-PAGE, the peak fractions were compiled and concentrated (100 kDa cut-off, Vivaspin 6, Sartorius).
10
Isolation and Analysis of the Gas1-GFP Protein Complex
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The native coimmunoprecipitation experiment was performed on enriched ER fractions as described (31 (link)). Briefly, 100 optical density at 600 nm (OD600) units of yeast cells were washed twice with TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, and protease inhibitor cocktail; Roche Diagnostics] and disrupted with glass beads, after which cell debris and glass beads were removed by centrifugation. The supernatant was then centrifuged at 17,000g for 15 min at 4°C. The pellet was resuspended in TNE, and digitonin was added to a final concentration of 1%. The suspension was incubated for 1 hour at 4°C with rotation, after which insoluble components were removed by centrifugation at 13,000g for 60 min at 4°C. For immunoprecipitation of Gas1-GFP, the sample was first preincubated with empty agarose beads (ChromoTek) at 4°C for 1 hour and subsequently incubated with GFP-Trap_A (ChromoTek) at 4°C for 3 hours. The immunoprecipitated beads were washed five times with TNE containing 0.2% digitonin, eluted with SDS sample buffer, resolved on SDS-PAGE, and analyzed by immunoblot.
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