His-ezrin proteins were produced in Rosetta (DE3). Briefly, the plasmids were transformed into E. coli strain, and protein expression was induced with 1 mM IPTG at 16 °C for 20 h. Then the protein was purified using Ni-NTA agarose (Qiagen) according the manufacturer’s instructions. Flag-ROCK2 were transfected into HEK293T cells and then enriched by FLAG-M2 resin (Sigma). All purification procedures were performed at 4 °C, and protease inhibitor cocktail (Sigma) was added to prevent protein degradation. All purified proteins were analyzed and confirmed with SDS/PAGE.
Flag m2 resin
Manufactured by Merck Group
The FLAG M2 resin is a laboratory equipment product used for affinity purification of recombinant proteins. It is designed to bind and capture proteins tagged with the FLAG peptide sequence. The resin provides a reliable and efficient method for the isolation and purification of FLAG-tagged proteins from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (5 mentions)
G coated sepharose beads, by GE Healthcare (2 mentions)
Ha resin, by Roche (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Aicar, by LGC (1 mentions)
Sakamototide, by GL Biochem (1 mentions)
C2c12 cell line, by Merck Group (1 mentions)
A769662, by Selleck Chemicals (1 mentions)
Protein g sepharose, by GE Healthcare (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Anti arnt2, by Santa Cruz Biotechnology (1 mentions)
Anti flag, by Merck Group (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
N ethylmaleimide, by Merck Group (1 mentions)
Gfp trap a, by Proteintech (1 mentions)
Glyceryl trioleate, by Merck Group (1 mentions)
24 protocols using flag m2 resin
1
Purification of His-ezrin and Flag-ROCK2 Proteins
2
Studying AMPK Regulation and Signaling
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AICAR was from Toronto Research Chemicals. A769662 was from Selleck Chemicals. Compound 13 was obtained as previously described (18 (link)). 991 (5-{[6-chloro-5-(1-methylindol-5-yl)-1H-benzimidazol-2-yl]oxy}-2-methyl-benzoic acid; CAS no. 129739-36-2) as obtained as previously described (7 (link)). Protein G Sepharose was from GE Healthcare, and FLAG-M2 resin from Sigma-Aldrich. ECL reagent and P81 filter papers were obtained from GE Healthcare. [γ-32P]-ATP was from Perkin-Elmer. AMARA, LKBtide, and Sakamototide substrate peptides were synthesised by GL Biochem. The COS1 cell line was obtained from American Type Culture Collection, and the C2C12 cell line was obtained from Sigma-Aldrich. Frozen tissues or extracts from AMPKα1-/α2-, AMPKβ1-/β2-, and AMPKγ3-deficient mice were obtained from Benoit Viollet and Marc Foretz (Institut National de la Santé et de la Recherche Médicale, Institut Cochin, Paris, France), Gregory Steinberg (McMaster University, Hamilton, ON, Canada), and Alexander Chibalin and Juleen Zierath (Karolinska Institutet, Stockholm, Sweden), respectively. All cell culture reagents were purchased from Thermo Fisher Scientific, and all other chemicals were from Sigma-Aldrich unless otherwise stated.
3
Analyzing BAP1 and IP3R3 Binding
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Cells (fibroblasts cell cultures or HEK-293, as indicated in the Figures) were collected and lysed in buffer containing 30 mM Tris-HCl, at pH 7.5, 50 mM NaCl, 1% NP-40. To map the BAP1 and IP3R3 binding region, HEK-293 cells were transiently transfected using polyethylenimine, collected 24 hours later, and lysed in 50 mM Tris, at pH 7.5, 150 mM NaCl, glycerol 10%, 1 mM EDTA, 50 mM NaF and NP-40 0.1%. All the buffers were supplemented with proteases and phosphatases inhibitors. Extracted proteins were pre-cleared by incubating lysates with G-coated sepharose beads (GE Healthcare) for 1 hour at 4 °C, then the supernatant (700 μg, referred as “Input”) was incubated overnight with IP3R3 antibody at 4 °C; precipitation of the immune complexes was performed with G-coated sepharose beads for 4 hours at 4 °C, according to manufacturer's instructions. Alternatively, the supernatant was incubated for 3 hours at 4 °C with FLAG® M2 resin (Sigma, cat. no A2220) or HA resin (Roche, cat. no. 11815016001). After immunoprecipitation, the beads were washed three times with lysis buffer, at 4 °C, and suspended in 40 μl of 2X Laemmli buffer. 10-20 μl (depending on experiment) were loaded on the gel and the samples were processed by SDS-PAGE and analyzed by WB.
4
ARNT2 Immunoprecipitation Protocol
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For immunoprecipitations, cells were washed twice in PBS and lysed in 20 mM HEPES, pH 8.0, 420 mM NaCl, 0.5% Igepal, 25% glycerol, 0.2 mM EDTA, 1.5 mM MgCl2, 1 mM DTT and protease inhibitors (Sigma). 100 µg of protein whole cell extract was diluted to 1 mg/ml in immunoprecipitation (IP) buffer (20 mM HEPES, pH 8.0, 150 mM NaCl, 150 mM KCl, 0.1% glycerol, 1 mM EDTA, 1 mM DTT and protease inhibitors) and incubated with 50 µL of BSA blocked Flag M2 resin (Sigma). The resin was washed twice with 1 mL of IP wash buffer (250 mM NaCl, 20 mM HEPES pH 8.0, 0.1% Igepal, and 1 mM EDTA) and the bound material boiled in 20 µl of SDS sample buffer. Input sample (10%) and immunoprecipitations were then run on 7.5%SDS-PAGE gels and transferred to nitrocellulose. Lysates from reporter gene assays were separated on 7.5% SDS-PAGE gel and transferred to nitrocellulose. Proteins were detected using the anti-ARNT2 (Santa Cruz), anti-FLAG (Sigma), anti-Myc (4A6, Upstate) and anti-α-Tubulin antibodies (MCA78G, Serotec). Primary antibodies were detected using horseradish peroxidise-conjugated secondary antibodies and visualised using chemiluminescence. Quantification of ARNT2 co-immunoprecipition band intensity was estimated using ImageLab software (BioRad).
5
Analyzing BAP1 and IP3R3 Binding
6
Examining Ubiquitylation in HEK 293T Cells
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To examine ubiquitylation, HEK 293T cells were transfected using calcium chloride and/or infected with the indicated viruses, and 24 h later were lysed in TNE buffer (50 mM Tris/HCl, pH 7.5, 250 mM NaCl, 1 % NP-40, 1 mM EDTA and 1 mM DTT) supplemented with N-ethylmaleimide (Sigma-Aldrich) as described previously (Yamazaki et al., 2009 (link)). Cell lysates were centrifuged (10 000 g for 30 min) and cleared supernatants were mixed with 1 vol. 2 % SDS TNE. Samples were heated at 90 °C for 10 min to destroy all non-covalent interactions. Lysates were diluted 10-fold in TNE buffer and subjected to FLAG immunoprecipitation for 16 h using FLAG M2 resin (Sigma-Aldrich). Samples were washed three times in TNE buffer and finally analysed by immunoblotting.
7
Affinity Capture of Protein Complexes
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HEK293T cells or MDA-MB-231 cells were collected and lysed in IP buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, 0.2% Triton X-100, 1 mM DTT, 10% glycerol) supplemented with Protease Inhibitor Cocktail (Sigma). Cell lysates were clarified by centrifugation at 12000 rpm for 20 min at 4°C. For different purposes, clarified cell lysates were incubated with acetyl lysine antibody coupled agarose (Immunechem), GFP-Trap A (Chromotek), and FLAG-M2 resin (Sigma) for 4 h before washing, respectively. The binding fraction was washed with IP buffer five times before being resolved by SDS–PAGE and immunoblotted with indicated antibodies.
8
Immunoprecipitation of Cul-2 Complexes
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HEK293T cells were seeded in 10-cm-diameter dishes and transfected with 5 μg of the indicated plasmids using PEI. After 24 h cells, were lysed with phosphate-buffered saline (PBS) supplemented with 0.5% NP-40 and protease and phosphatase inhibitors (Roche). Cleared lysates were incubated with FLAG M2 resin (Sigma) for 16 h at 4°C. For the analysis of Cul-2 endogenous complexes, the cells were lysed in 50 mM Tris-HCl (pH 7.5)–250 mM NaCl–1% NP-40–1 mM EDTA–1 mM dithiothreitol (DTT). The next day, the beads were washed 3 times with lysis buffer prior to incubation at 95°C for 5 min in Laemmli loading buffer to elute bound proteins. Cleared lysates and FLAG eluates were analyzed by SDS-PAGE and immunoblotting. Data shown are representative of at least 3 independent experiments showing similar results.
9
Monitoring Autophagy Lipid Dynamics
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DOPE (Avanti Polar Lipids; 850725), DOPC (Avanti Polar Lipids; 850375), NBD-PS (Avanti Polar Lipids; 810198), Liss Rhod-PE (Avanti Polar Lipids; 810150), DGS-NTA(Ni) (Avanti Polar Lipids; 790404), PI4,5P2 (Avanti Polar Lipids; 840046), glyceryl trioleate (Sigma-Aldrich; T7140), BODIPY 558/568 C12 (Invitrogen; D3835), Imperial protein stain (Thermo Scientific; 24617), Flag M2 resin (Sigma-Aldrich; A2220), WIPI-4-TurboGFP (Origene; WDR45-tGFP transcript variant 1, RG209654), BSA-Oleate monounsaturated fatty acid complex and BSA control (Cayman Chemical; 29557 and 29556), rabbit anti-ATG2A (Cell Signalling; 15011), rabbit anti-ATG2B (Sigma-Aldrich; HPA019665), mouse anti-WIPI-4 (Santa Cruz Biotechnology; sc398272), rabbit anti-LC3B (D11) (Cell Signalling; 3868), rabbit anti-p62 (Cell Signalling; 95697), rabbit anti-Plin3 (Sigma-Aldrich; HPA006427), rabbit anti-beta-actin (Cell Signalling; 4970), mouse anti-Flag M2 (Millipore; F1804), rabbit anti-GFP (Cell Signalling; 2956), secondary antibodies, AlexaFluor 647 goat anti-mouse (Life Technologies; A21236), sodium dithionite Sigma-Aldrich (157953), DMEM (Gibco; 11965-092), MEM (Gibco; 31985-070), Expi293 medium (Gibco; A14351-01), 1x EBSS (Gibco; 24010-043).
10
Affinity Purification of Flag-Tagged Proteins
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HEK 293T cells were transfected with pcDNA4/TO, pcDNA4/TO-UBCv1, or pcDNA4/TO-UBCv1C85A using polyethylenimine (PEI) or Lipofectamine 2000 (LF2000) transfection reagent and following manufacturer’s instructions. After 24 h, cells were washed once with ice-cold PBS and lysed with IP buffer [10% glycerol, 10 mM CaCl2, 150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 0.1% Triton-X100, and proteases/phosphatases inhibitors (Roche)]. After centrifugation (15,000 × g for 20 min), supernatants were incubated with Flag M2 resin (Sigma Aldrich) at 4°C for 16 h. After three washes with ice-cold IP buffer, beads were boiled and analyzed by WB.
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