N‐terminal Flag‐tagged full‐length DDX21 and its mutants or truncated DDX21 helicase core and C‐terminal HA‐tagged full‐length NS1 were cotransfected into HEK293T cells. Cells were collected after 48 h post‐transfection and lysed in a buffer consisting of 25 × 10−3m Tris‐HCl 7.5, 150 × 10−3m NaCl, 5% glycerol, 0.1 × 10−3m DTT supplemented with complete EDTA‐free protease inhibitor cocktail tablets (Roche, Basel, Switzerland). After centrifugation, the cell supernatants were used for co‐IP assay with anti‐Flag beads (Sigma‐Aldrich, Saint Louis, USA) and the elutes from anti‐Flag beads were then loaded to SDS‐PAGE and transferred to a nitrocellulose membrane for sequential immunoblotting analysis with anti‐Flag M2‐HRP or anti‐HA‐HRP antibodies Sigma‐Aldrich, Saint Louis, USA).
Anti flag beads
Manufactured by Merck Group
Sourced in United States
Anti-FLAG beads are a type of affinity resin used for the purification and detection of proteins tagged with the FLAG epitope. They consist of agarose beads coated with a high-affinity anti-FLAG monoclonal antibody. The beads can capture and isolate FLAG-tagged proteins from complex samples, enabling their subsequent analysis and characterization.
Automatically generated - may contain errors
Lab products found in correlation
Flag peptide, by Merck Group (15 mentions)
Anti flag, by Merck Group (12 mentions)
Anti ha, by Merck Group (7 mentions)
Anti ha beads, by Merck Group (6 mentions)
Odyssey imaging system, by LI COR (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (4 mentions)
Edta free protease inhibitor cocktail, by Roche (4 mentions)
Tgx precast gel, by Bio-Rad (3 mentions)
Anti ha beads, by Santa Cruz Biotechnology (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (3 mentions)
Protein a g bead, by Santa Cruz Biotechnology (3 mentions)
3xflag peptide, by Merck Group (3 mentions)
Anti rabbit igg hrp, by Merck Group (3 mentions)
Anti mouse igg peroxidase, by Merck Group (3 mentions)
β actin, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (2 mentions)
Fluorescently conjugated antibodies, by BioLegend (2 mentions)
153 protocols using anti flag beads
1
Co-IP of DDX21 and NS1 proteins
2
Immunoprecipitation and Western Blotting Protocol
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Tissues were lysed in RIPA buffer containing proteinase inhibitors, protein phosphatase inhibitors and an OGA inhibitor. For immunoprecipitation, whole-cell lysates were incubated with and precipitated by anti-Flag beads (Millipore Sigma). Equal amounts of whole lysates or immunoprecipitation samples were electrophoresed on TGX precast gels (Bio-Rad) and transferred to nitrocellulose membrane. Membranes were incubated with primary antibodies at 4°C for overnight. Western blotting was visualized by using IRDye secondary antibodies and the Odyssey imaging system (LI-COR Biosciences).
3
Immunoprecipitation and Western Blotting Protocol
4
Flag-tagged Protein Immunoprecipitation and Analysis
5
Co-purification of Protein Complexes
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BL21 (DE3) E. coli cells were co-transformed with both pET17-twin-strep-C16 and pET28b-flag-C4 and grown in standard LB medium supplemented with ampicillin and kanamycin. Protein expression was induced upon the addition of IPTG at 0.5 mM final concentration once cells had reached an optical density (600 nm) of 0.8–1.0 and the temperature had been reduced from 37 to 16 °C. After growth overnight, cells were pelleted by centrifugation and then resuspended in buffer A (50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 5% (w/v) glycerol, 0.5 mM TCEP), supplemented with lysozyme, benzonase nuclease (Merck Life Sciences) and cOmpleteTM EDTA-free protease inhibitor (Roche). Cells were lysed by sonication and cleared by centrifugation.
Pull-downs were performed using the cell lysate from the C16 and C4 co-expression. The lysate was divided in half, and then applied to either Strep-Tactin beads (IBA, catalog number 2-1613-002, 1 ml to obtain a bed volume of 50 µl) or Anti-Flag beads (Merck, catalog number M8823, 100 µl to obtain a bed volume of 50 µl). After extensive washing, proteins retained on the beads were eluted with the addition of either biotin or flag peptide respectively and the eluted proteins analysed by SDS-PAGE and immunoblotting with either anti-strep (Merck, catalog number SAB27002216, 1:7000 dilution) or anti-flag antibodies (Merck, catalog number F3165, 1:5000 dilution).
Pull-downs were performed using the cell lysate from the C16 and C4 co-expression. The lysate was divided in half, and then applied to either Strep-Tactin beads (IBA, catalog number 2-1613-002, 1 ml to obtain a bed volume of 50 µl) or Anti-Flag beads (Merck, catalog number M8823, 100 µl to obtain a bed volume of 50 µl). After extensive washing, proteins retained on the beads were eluted with the addition of either biotin or flag peptide respectively and the eluted proteins analysed by SDS-PAGE and immunoblotting with either anti-strep (Merck, catalog number SAB27002216, 1:7000 dilution) or anti-flag antibodies (Merck, catalog number F3165, 1:5000 dilution).
6
Semi-in vitro Ubiquitination Assay
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The semi-in vitro ubiquitination assay was performed as described previously (Wang et al. 2013) (link). HIS-GCN20 coupled to Ni-IDA Beads (Smart-Lifesciences, SA003025) was incubated with protein extracts of Col-0 or wee1 in ubiquitination buffer (25 mm Tris-HCl, pH 7.5, 10 mm MgCl 2 , 10 mm NaCl, 10 mm ATP, 5 mm DTT, and 100 µm MG132). After 4 h of incubation, the beads were washed 3 times with RIPA buffer containing 500 mm NaCl and boiled in 2 × SDS loading buffer at 100 °C for 8 min, followed by immunoblotting using anti-ubiquitin antibody (1:1,000, Cell Signaling Technology, 3933).
In vivo ubiquitination assays GCN20-FLAG was expressed in Col-0 or wee1 protoplasts as described previously (Yoo et al. 2007 (link)). After 12 h of incubation in the absence or presence of 5 mm HU, total proteins were extracted from the samples using extraction buffer (100 mm Tris-HCl, pH 7.0, 150 mm NaCl, 0.1% Nonidet P-40, 1 mm PMSF, 100 μm MG132, and protease inhibitor cocktail) and precipitated by anti-FLAG beads (Merck, M8823), followed by immunoblotting using anti-ubiquitin (1:1,000, Cell Signaling Technology, 3933), anti-FLAG (1:4,000, Sigma-Aldrich, A8593), and anti-actin (1:2,000, Agrisera, AS132640) antibodies.
In vivo ubiquitination assays GCN20-FLAG was expressed in Col-0 or wee1 protoplasts as described previously (Yoo et al. 2007 (link)). After 12 h of incubation in the absence or presence of 5 mm HU, total proteins were extracted from the samples using extraction buffer (100 mm Tris-HCl, pH 7.0, 150 mm NaCl, 0.1% Nonidet P-40, 1 mm PMSF, 100 μm MG132, and protease inhibitor cocktail) and precipitated by anti-FLAG beads (Merck, M8823), followed by immunoblotting using anti-ubiquitin (1:1,000, Cell Signaling Technology, 3933), anti-FLAG (1:4,000, Sigma-Aldrich, A8593), and anti-actin (1:2,000, Agrisera, AS132640) antibodies.
7
Phosphorylation analysis of GCN20-FLAG
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The GCN20-FLAG plasmid was transfected into Col-0 or wee1 protoplasts. After 8 h of incubation, the protoplasts were treated with 5 mm HU for 8 h. Total proteins were extracted from the samples using immunoprecipitation buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.1% Nonidet P-40, 0.1% Triton X-100, 100 μm ATP, 1 mm PMSF, protease inhibitor cocktail, PhosSTOP Phosphatase Inhibitor Cocktail, and 100 μm MG132). GCN20-FLAG was immunoprecipitated using anti-FLAG beads (Merck, M8823) and subjected to SDS-PAGE analysis using gels with or without 40 μm Phos-Tag (Wako, 300-93523), followed by immunoblotting using anti-FLAG (1:4,000, Sigma-Aldrich, A8593) antibody.
8
Immunoprecipitation of Flag-USP3 and HA-Aurora A
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The ECa109 cells expressing Flag-USP3 or HA-Aurora A were first washed on a 6 cm culture plate with PBS buffer. The cells on each plate were then scraped off and were lysed with 1 ml of ice-cold RIPA lysis buffer. Cell lysates were collected and anti-Flag beads (Sigma, USA) or anti-HA beads (Sigma, USA) were added. The resulting immunoprecipitate was shaken overnight at 4 °C. The beads were washed and loaded into the loading buffer, and the levels of bound proteins were detected by SDS-PAGE followed by western blotting.
9
Phosphorylation of TLE6-S510Y Protein
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Wild-type and TLE6-S510Y proteins were expressed in HEK293 and immunoprecipitated using anti-Flag beads (Sigma-Aldrich, St. Louis, MO, USA). A total of 20 μg of purified proteins was added to a standard PKA mixture containing 20 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM unlabeled ATP, 1 mM 1,4-dithiothreitol, 100 mM NaCl, and 1 mM [γ32P]ATP (0.5 Ci/mmol- Perkin Elmer, Waltham, MA, USA), with different amounts of PKA (Promega, Madison, WI, USA) as indicated and incubated for 1 h at 37 °C. Reactions were stopped by adding 20 μL of 2X Laemmli sample buffer and proteins were separated by SDS-PAGE electrophoresis followed by autoradiography and immunoblotting.
10
Purification of Heterologous Proteins
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tsA 201 cells, which are SV40-transformed HEK 293 cells, were used because they express heterologous proteins at high levels. Cells were grown in 162-cm2 flasks and transfected using calcium phosphate. Pretreatments with (+)SKF10047 and BD1047 were for 2 h at 37°C, and stimulation with 5 µM thapsigargin was for 30 min at 20°C. Cells were extracted in 25 ml of ice-cold medium, and all subsequent steps were performed at 4°C. The suspension was centrifuged at 1,000 g for 5 min, and pelleted cells were solubilized for 60 min in 500 µl TS. After centrifugation (50,000 g for 60 min), 50 µl of the supernatant was removed for analysis of total expression (input), and 450 µl was incubated with 30 µl anti-Myc (EZ View Red) or anti-FLAG beads (Sigma-Aldrich) for 3 h with rotation. Protein–bead complexes were isolated (20,800 g for 10 min) and washed three times in TS, and proteins were eluted either with 50 µl of the peptides (1 mg/ml; Sigma-Aldrich), to which the anti-Myc or anti-FLAG antibodies had been raised, or with 50 µl Laemmli buffer. The eluted samples were analyzed by SDS-PAGE followed by immunoblotting. For immunoblots, lanes were loaded with 10 µl of the 500-µl sample (2% of the entire sample) for the measurement of input and with 10 or 20 µl of the 50-µl eluate for measurements of immunoprecipitation.
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