For Chromatin IP (ChIP), a previously described protocol was used (Lopez et al. 2017 (link)) with the following modifications. Anti-FLAG antibody-conjugated beads (Cat# A8592; Sigma, were used in pull-down. Ct values for each ChIP samples were first normalized to the corresponding input samples, and then divided by the values for the CAN1 locus to calculate the relative fold enrichment. Primers used in the qPCR analysis were previously described (Lopez et al. 2017 (link)). P-values were calculated using the Student t-test. For the TMPyP4 ChIP, the cells were grown at 30° in liquid YPD containing 50 µM TmPyP4. Next day, they were diluted in liquid YPD containing 50 µM TmPyP4 and grown till mid-log phase (O.D600 0.7–0.8). Cells were cross-linked and further processed as above.
Anti flag antibody conjugated beads
Manufactured by Merck Group
Anti-FLAG antibody-conjugated beads are a laboratory tool used for the purification and detection of proteins containing a FLAG tag. The beads are coated with a specific antibody that binds to the FLAG tag sequence, allowing for the efficient capture and isolation of the tagged proteins from complex samples.
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Lab products found in correlation
Protease inhibitor cocktail, by Roche (4 mentions)
Ubiquitin, by Santa Cruz Biotechnology (1 mentions)
Hsp90, by Enzo Life Sciences (1 mentions)
Flag tag (flag), by Thermo Fisher Scientific (1 mentions)
Gapdh, by Enzo Life Sciences (1 mentions)
Phosstop, by Roche (1 mentions)
Ecl assay kit, by GE Healthcare (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Anti ha antibody conjugated beads, by Merck Group (1 mentions)
Transit lt1 reagent, by Mirus Bio (1 mentions)
Nupage 4 to 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Flag peptide, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Protein g beads, by GenScript (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
14 protocols using anti flag antibody conjugated beads
1
ChIP Assay with Anti-FLAG Antibody
2
Immunoprecipitation and Immunoblotting of VHL-HIF1α Complexes
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Proteins were extracted from transiently transfected human embryonic kidney (HEK293) as previously described. (Woodford et al. 2016 (link)) For immunoprecipitation, cell lysates were incubated with anti-FLAG antibody conjugated beads (Sigma) for 2 h at 4ºC. Immunopellets were washed 3 times with lysis buffer (20 mM HEPES (pH7.0), 100-mM NaCl, 1-mM MgCl2, 0.1% NP40, protease inhibitor cocktail (Roche), and PhosSTOP (Roche)). Precipitated proteins were resuspended in 5X Laemmli buffer, boiled, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Co-immunoprecipitated proteins were detected by immunoblotting. Immunoblotting was performed with the indicated antibodies recognizing VHL, HA, HIF1α, human Mps1 (TTK) (Cell Signaling), Ubiquitin (Santa Cruz Biotechnology), Hsp90, GAPDH (ENZO Life Sciences), 6x-His, and FLAG (Thermo Fisher Scientific).
3
FLAG-tagged Protein Immunoprecipitation
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HEK293 cells were maintained in DMEM in the presence of 10% fetal bovine serum (FBS) and penicillin/streptomycin. A day before transfection, cells were plated in 60 mm dishes at 1 × 106 cells/dish. The relevant plasmids were transfected with Lipofectamine 2000 transfection reagent (Invitrogen), in accordance with the manufacturer’s instructions. After 36 h, cells were washed with ice-cold PBS and solubilized with 1 ml lysis buffer, containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail, for 30 min on ice. The lysates were centrifuged at 15,000 rpm for 10 min at 4°C, then supernatants were incubated with anti-FLAG antibody-conjugated beads (Sigma) at 4°C for 2 h. The beads were washed four times with lysis buffer. Bound proteins were eluted by boiling in sodium dodecyl sulfate (SDS) sample buffer and separated by SDS-polyacrylamide gel electrophoreses (SDS-PAGE). To examine the expression of specific proteins, 20 μg clarified lysate was loaded into each well of the gel. Proteins were transferred onto a nitrocellulose membrane and probed with the relevant antibodies, and then detected using the ECL assay kit (GE Healthcare Life Science, USA).
4
Immunoprecipitation and Western Blot Analysis
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Cells were lysed in lysis buffer containing 1% NP-40, 10% glycerol, 135 mM NaCl, and 20 mM Tris–HCl, pH 8.0, supplemented with 1× protease inhibitor cocktails (Roche). Whole cell lysates were pre-cleared with isotype IgG and Protein G Dynabeads™ (Life Technologies, Oslo, Norway). FLAG-TET2 was then immunoprecipitated from the pre-cleared whole cell lysate with anti-FLAG antibody-conjugated beads (Sigma-Aldrich, Saint Louis, MO) and washed with lysis buffer four times. For RUNX1 immunoprecipitation, the pre-cleared whole cell lysates were incubated with 4 µg anti-RUNX1 antibody (for control groups, 4 µg isotype IgG was added) overnight and then incubated with 40 µl Protein-G Dynabeads™ for 2 h and washed with lysis buffer four times. The beads were boiled directly with 1× SDS loading buffer. Precipitates were subjected to SDS–PAGE and Western blotting was performed using indicated antibodies. Antibodies used in this study were listed in Table S11 .
5
Ago2-Bound RNA Characterization
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Cultured cortical neurons grown on 10 cm dishes were transduced with lentivirus expressing Flag-tagged Ago2 at DIV 7, and treated with NMDA at DIV 17. Cross-linking and immunoprecipitation of Ago2-associated RNA was performed as described with modifications 70 (link). At 90min after NMDA treatment, neurons were crosslinked with UV irradiation (480,000 µJ c−1m−2), and then lysed in 1XRIPA buffer containing 0.1 U µL−1 RNase inhibitor followed by sonication. After centrifugation at 13,000 rpm for 30 min at 4°C, supernatant was incubated with anti-Flag antibody-conjugated beads (Sigma-aldrich) overnight with rotation at 4°C. The beads were washed 5 times with immunoprecipitation buffer [25mM HEPES (PH7.5), 150mM NaCl, 0.5mM EDTA, 1mM EGTA, 10% Glycerol, 0.1% NP40, 1mM NaF, 1mM 2-glycerophosphate, 1mM Na3VO4]. Bound RNAs were extracted with phenol/chloroform, precipitated with ethanol, and analyzed by qRT-PCR.
6
Detecting Protein Modifications in HeLa Cells
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HeLa cells at 70–80% confluency, transfected with the indicated plasmids or treated with H2O2, were washed with 1 × phosphate-buffered saline (PBS) and resuspended in RIPA buffer (1 × PBS, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) along with 1 × protease inhibitor cocktail and phosphatases inhibitor cocktail (Roche Applied Science). To detect PML SUMOylation in HeLa cells, whole-cell extracts in the presence of NEM were prepared. Lysed cells were centrifuged at 4°C at 12 000 r.p.m. for 10 min, and the supernatant was incubated with protein A-conjugated beads for preclearing. To detect protein–protein interactions or acetylation and SUMO1 modification of PML, whole-cell extracts were incubated with anti-HA antibody-conjugated beads (Sigma F2426) or anti-FLAG antibody-conjugated beads (Sigma E6779) for 2 h. The beads were washed with NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, and 0.1% NP-40) five times, and supernatants were discarded. Then, 2 × sample buffer was added to the beads, followed by SDS-PAGE and western blotting as previously described.9 (link), 10 (link)
7
NMDA-induced Ago2 CLIP in Neurons
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Cultured cortical neurons grown on 10-cm plates were transduced with lentivirus expressing Flag-tagged Ago2 at 7 DIV and treated with NMDA at 17 DIV. Cross-linking and immunoprecipitation of Ago2-associated RNAs was performed as described previously (Hu et al., 2014 (link)). At 90 min after NMDA treatment, neurons were cross-linked by UV irradiation (480,000 µJ/cm2), and then lysed in 1× RIPA buffer containing 0.1 U/µl RNase inhibitor followed by sonication. After centrifugation at 13,000 rpm for 30 min at 4°C, supernatant was incubated (overnight at 4°C, with rotation) with anti-Flag antibody–conjugated beads (Sigma-Aldrich) in the immunoprecipitation buffer (25 mM Hepes, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1 mM EGTA, 10% glycerol, 0.1% NP-40, 1 mM NaF, 1 mM 2-glycerophosphate, and 1 mM Na3VO4). The beads were washed five times with the immunoprecipitation buffer. Bound RNAs were extracted with phenol/chloroform, precipitated with ethanol, and analyzed by qRT-PCR.
8
Flag-tagged Ash2l-mutant Immunoprecipitation
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Flag-tagged Ash2l-mutant variants encoding plasmid were transfected into the ESCs and subjected to immunoprecipitation experiments. The ESCs were transfected with 3 μg plasmid by using TransIT-LT1 reagent (Mirus) and disrupted with immunoprecipitation lysis buffer. The lysate was incubated with anti-Flag antibody-conjugated beads (A2220, Sigma-Aldrich) for 2 h at 4°C. Beads were then washed three times by IP buffer and eluted by boiling in 20 μl SDS-containing protein loading buffer. Twenty microliters of eluted samples were individually assigned for western blot with 5% input. Flag-tagged bead without peptide conjugation was used as a negative control.
9
Affinity Purification of Ago2-Bound RNAs
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Cultured cortical neurons (DIV7) were transduced with lentivirus-expressing Flag-tagged Ago2. At 10 days after transduction, neurons were crosslinked with ultraviolet irradiation (480,000 μJ c−1 m−2) and then lysed in RIPA buffer containing 0.1 U μl−1 RNase inhibitor followed by sonication. After centrifugation at 13,000 r.p.m. for 30 min at 4 °C, the supernatant was incubated with anti-Flag antibody-conjugated beads (Sigma-Aldrich) overnight with rotation at 4 °C. The beads were washed five times with immunoprecipitation buffer (25 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 1 mM EGTA, 10% glycerol, 0.1% NP40, 1 mM NaF, 1 mM 2-glycerophosphate and 1 mM Na3VO4). Bound RNAs were extracted with phenol/chloroform, precipitated with ethanol and analysed by qRT–PCR.
10
FLAG-ASXL1 Immunoprecipitation and Mass Spectrometry
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IP was performed using nuclear fraction buffer and washed with IP buffer [20 mM tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 mM NaF, and protease inhibitor cocktail (Roche)] for four times. For FLAG-tagged ASXL1, the IPs were eluted with 3×FLAG peptide (100 ng/ml; Sigma-Aldrich, F4799) in phosphate-buffered saline for 30 min at room temperature. All the IPs were performed with nuclear extraction.
FLAG-ASXL1 was immunoprecipitated from the nuclear extracts with anti-FLAG antibody–conjugated beads (Sigma-Aldrich), and the associated proteins were eluted from the beads by FLAG peptides. The eluates were then resolved on NuPAGE 4 to 12% Bis-Tris Gel (Invitrogen) followed by Coomassie brilliant blue staining, and lanes were excised for MS analysis by the Taplin Biological Mass Spectrometry Facility (Harvard Medical School).
FLAG-ASXL1 was immunoprecipitated from the nuclear extracts with anti-FLAG antibody–conjugated beads (Sigma-Aldrich), and the associated proteins were eluted from the beads by FLAG peptides. The eluates were then resolved on NuPAGE 4 to 12% Bis-Tris Gel (Invitrogen) followed by Coomassie brilliant blue staining, and lanes were excised for MS analysis by the Taplin Biological Mass Spectrometry Facility (Harvard Medical School).
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