To examine ubiquitination of ectopically expressed IFNAR1, 293T cells expressing ACE2/TMPRSS2 were transfected with FLAG-IFNAR1 and HA-ubiquitin for 36 h and then infected with SARS-CoV-2 for 12 h or treated with thapsigargin (2 μM) for 30 min. The cells were lysed in 1% SDS followed by quenching of SDS with a 10-fold excess of Triton X-100. Five hundred micrograms of total protein was used for immunoprecipitation with anti-FLAG M2 agarose beads (number A2220; Sigma) at 4°C for 2 h, and IFNAR1 ubiquitination was assessed by Western blotting with an anti-HA antibody (number F7425; Sigma). To assess ubiquitination of endogenous IFNAR1, ACE2/TMPRSS2-expressing 293T cells were transfected with FLAG-tagged TR-TUBE for 36 h, followed by infection with SARS-CoV-2 for 12 h and as a positive control with VSV (MOI of 0.01) for 12 h. The cells were harvested in lysis buffer containing 0.25% NP-40, and 500 μg of total protein was subjected to immunoprecipitation with anti-FLAG M2 agarose beads (Sigma) followed by detection of polyubiquitinated IFNAR1 by Western blotting with an anti-IFNAR1 antibody.
Anti flag m2 agarose bead
Manufactured by Merck Group
Sourced in United States, Germany
Anti-FLAG M2 agarose beads are solid-state agarose beads conjugated with the anti-FLAG M2 monoclonal antibody. They are designed for the purification and detection of recombinant proteins tagged with the FLAG peptide sequence.
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Lab products found in correlation
Flag peptide, by Merck Group (37 mentions)
Protease inhibitor cocktail, by Roche (11 mentions)
3xflag peptide, by Merck Group (9 mentions)
Protease inhibitor cocktail, by Merck Group (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Complete protease inhibitor cocktail, by Roche (4 mentions)
Polyclonal anti flag, by Merck Group (4 mentions)
Anti ha agarose beads, by Merck Group (4 mentions)
Protease inhibitor, by Roche (4 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (4 mentions)
Ni2 nta agarose bead, by Qiagen (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Gfp trap beads, by Proteintech (3 mentions)
Mobicol spin column, by MoBiTec (3 mentions)
Complete protease inhibitor mixture, by Roche (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (3 mentions)
Anti ha, by Merck Group (3 mentions)
Ni nta agarose, by Qiagen (3 mentions)
Anti α tubulin, by Merck Group (3 mentions)
Kinase buffer, by Cell Signaling Technology (3 mentions)
261 protocols using anti flag m2 agarose bead
1
Ubiquitination of IFNAR1 in SARS-CoV-2 Infection
2
Isolation of FLAG-tagged CARF Complexes
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TOCARF cells were cultured to 80% confluency in four 150-mm cell culture dishes in normal medium with or without 1 ng/ml doxycycline for 48 h. After the cells were washed with phosphate buffer [PBS(−)], they were lysed in buffer A (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.5% IGEPAL CA-630; 1 mM Na3VO4; and 1 mM PMSF) and incubated on ice for 30 min. The cell extracts were obtained as the supernatant following centrifugation of the lysate at 4°C for 30 min at 22,180 × g. Cell extracts (15 mg) were incubated with anti-FLAG M2 agarose beads (Sigma) for 4 h at 4°C. The beads were then washed five times in buffer A and then once with buffer B (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1 mM Na3VO4; and 1 mM PMSF). The FLAG–CARF-associated complexes were released from the anti-FLAG M2 agarose beads by adding 40 μl of 0.5 mg/ml FLAG peptide (Sigma) on ice for 30 min twice.
3
FLAG-tagged Protein Purification from Ovaries
4
Purification of KAT2A-containing HAT Complex
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Recombinant FLAG tagged KAT2A was purified using anti-FLAG M2-agarose beads (Sigma). M2-agarose-bound protein complexes were washed three times with immunoprecipitation (IP) buffer (25 mM Tris-HCl, pH 7.9, 10% (v/v) glycerol, 0.1% Nonidet P-40, 0.5 mM DTT, 5 mM MgCl2) containing 0.5 M KCl and twice with IP buffer containing 100 mM KCl. After washing, proteins were eluted with a 1000 × excess of the flag epitope peptide. The reconstituted human HAT module of the ATAC complex (rHAT-ATAC) containing the KAT2A, ADA2a, ADA3, and SGF29 subunits was expressed and purified from insect cells25 (link).
5
Drosophila S2 Cell Transfection and Immunoprecipitation
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Drosophila Schneider cells (S2 cells) were cultured in Shields and Sang M3 insect medium (Sigma) supplemented with 10% FCS and antibiotics at 25°C. Plasmids were transfected using Effectene (QIAGEN). To disrupt microtubule cytoskeletons, S2 cells were treated with 10 μM cholchicine. To suppress dynein function, S2 cells were treated with 10 μM ciliobrevin D. Cells were transfected with tubulin-GAL4 to drive various UAS constructs: UAS-ik2-HA, UAS-ik2-FLAG, UAS-ik2-G250D-HA, UAS-spn-F-myc, UAS-spn-F-FLAG, UAS-spnF-8A-myc, UAS-spnF-8D-myc, UAS-spnF-N-myc, UAS-spnF-C-myc, UAS-spnF-ΔCC1-myc, UAS-spnF-ΔCC2-myc, UAS-spnF-ΔCC3-myc, UAS-spnF-ΔSCD-myc, UAS-GFP-myc, and UAS-ctp-FLAG. Cells were lysed in lysis buffer (20 mM Tris pH 7.0, 150 mM NaCl, 2 mM EDTA, 1% TritonX-100, 1 mM DTT and protease inhibitors (Roche)). The cell lysates were incubated with anti-FLAG M2 agarose beads (Sigma) on ice and washed thoroughly with lysis buffer. Proteins were eluted and detected using immunoblot assays with rat antibody to HA (Roche), rabbit antibody to Myc (Santa Cruz Biotechnology), mouse antibody to FLAG (Sigma), and appropriate HRP-conjugated secondary antibodies (Jackson Immuno Research).
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Drosophila Schneider cells (S2 cells) were cultured in Shields and Sang M3 insect medium (Sigma) supplemented with 10% FCS and antibiotics at 25°C. Plasmids were transfected using Effectene (QIAGEN). To disrupt microtubule cytoskeletons, S2 cells were treated with 10 μM cholchicine. To suppress dynein function, S2 cells were treated with 10 μM ciliobrevin D. Cells were transfected with tubulin-GAL4 to drive various UAS constructs: UAS-ik2-HA, UAS-ik2-FLAG, UAS-ik2-G250D-HA, UAS-spn-F-myc, UAS-spn-F-FLAG, UAS-spnF-8A-myc, UAS-spnF-8D-myc, UAS-spnF-N-myc, UAS-spnF-C-myc, UAS-spnF-ΔCC1-myc, UAS-spnF-ΔCC2-myc, UAS-spnF-ΔCC3-myc, UAS-spnF-ΔSCD-myc, UAS-GFP-myc, and UAS-ctp-FLAG. Cells were lysed in lysis buffer (20 mM Tris pH 7.0, 150 mM NaCl, 2 mM EDTA, 1% TritonX-100, 1 mM DTT and protease inhibitors (Roche)). The cell lysates were incubated with anti-FLAG M2 agarose beads (Sigma) on ice and washed thoroughly with lysis buffer. Proteins were eluted and detected using immunoblot assays with rat antibody to HA (Roche), rabbit antibody to Myc (Santa Cruz Biotechnology), mouse antibody to FLAG (Sigma), and appropriate HRP-conjugated secondary antibodies (Jackson Immuno Research).
6
Immunoprecipitation of FLAG-Tagged Argonaute Proteins from Chlamydomonas reinhardtii
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C. reinhardtii strains, expressing FLAG-AGO3 or FLAG-Ble, were grown to midlogarithmic phase in TAP medium, and ∼4 × 109 cells were pelleted by centrifugation and resuspended in 10 mL of lysis buffer (50 mM Tris⋅HCl, pH 7.5, 120 mM NaCl, 2 mM benzamidine, 0.2 mM PMSF, and 10% glycerol) containing 5 μL/mL plant protease inhibitor mixture (Sigma). From this step on, cells and lysates were always kept on ice. Cells were broken by 2 passages through a French press at a pressure of ∼2,000 psi. Cell lysates were then centrifuged at 16,000 × g for 30 min. CHAPS (0.1% final concentration) was added to the supernatant and an aliquot (∼10%) was saved for input control analyses of proteins and RNAs. The remaining supernatant was incubated with buffer-equilibrated anti–FLAG-M2 agarose beads (Sigma) for 2 h, and then the beads were washed 5 times with lysis buffer containing 0.1% CHAPS and 5 μL/mL plant protease inhibitor mixture. Proteins/RNAs associated with the beads were eluted by incubation with lysis buffer (without glycerol) containing 150 μg/mL 3×FLAG peptide (Sigma). RNA was purified from the eluate, precipitated with glycogen as carrier, and used for RT-PCR analyses (55 ).
7
Purification of Flag-Tagged Proteins
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The Flp-In T-REx 293 cells containing G5-WT-HEF or its point mutants were induced for expression with 1 µg/mL doxycycline for 48 h in 150-mm dishes. The cells were then harvested, washed once with PBS, lysed by vigorous mixing for 30 sec in 1 mL of lysis buffer (50 mM Tris-HCl at pH 8.0 containing 150 mM NaCl, 0.5% [w/v] IGEPAL-CA630, 1 mM PMSF), and incubated for 20 min on ice. Soluble whole-cell lysates were prepared by centrifugation at 20,000g for 20 min at 4°C and incubated with 10–15 µL of anti-Flag M2 agarose beads (Sigma-Aldrich) for 3 h at 4°C with rotation. The beads were washed five times with 1 mL of lysis buffer and once with 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, and then RNA–protein complexes (RNPs) were eluted from the beads with 500 µg/mL Flag peptide in the same buffer.
8
Kinase Activity Assay for ERK and MSK1
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For ERK kinase activity, GST or GST–Trim7 or Elk1 was incubated with ERK2 in buffer K (50 mM Tris-Cl pH 8.0, 150 mM NaCl, 10 mM MgCl2, 1mM EDTA and 2mM DTT) containing 0.2 mM ATP and 20 μCi γ32P ATP for 1 hr at 30 °C. The reaction was stopped by adding SDS loading dye and analysed on SDS PAGE. The gel was dried and bands were visualised by autoradiography.
For MSK1 kinase reaction, FLAG–MSK1 WT or FLAG–MSK1 D565A were ectopically expressed in 293T cells, immunoprecipitated by anti-FLAG M2 agarose beads (Sigma), washed and eluted using 100 μg ml−1 3X FLAG peptide (Sigma). Eluted MSK1 or MSK1 D565A was incubated with GST–Trim7 or GST–CREB in buffer H containing 20uM ATP and 20 μCi γ32P ATP for 1 hr at 30 °C. The reactions were analysed as described above.
For MSK1 kinase reaction, FLAG–MSK1 WT or FLAG–MSK1 D565A were ectopically expressed in 293T cells, immunoprecipitated by anti-FLAG M2 agarose beads (Sigma), washed and eluted using 100 μg ml−1 3X FLAG peptide (Sigma). Eluted MSK1 or MSK1 D565A was incubated with GST–Trim7 or GST–CREB in buffer H containing 20uM ATP and 20 μCi γ32P ATP for 1 hr at 30 °C. The reactions were analysed as described above.
9
Protein-Protein Interaction Detection
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FLAG-mANGPTL8 and Pe-CFP-PIRB were transfected into 293-T cells using Lipo8000™ Transfection Reagent. After 24 h, 293-T cells were collected and lysed in ice-cold IP buffer (containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P40 (Np40), proteinase inhibitor (Roche), 0.1% SDS-Na) for 30 min. The lysates were sonicated and centrifuged at 13,000 × g for 15 min at 4 °C. The supernatants were collected and incubated with anti-FLAG-M2 agarose beads (Sigma) overnight at 4 °C. The beads were rinsed with ice-cold IP buffer three times and then heated at 95 °C in loading buffer, followed by western blot analyses. Images of the precipitated proteins were captured by a Bio-Rad imaging system and analyzed by Image J.
10
ANKRD1 Protein Interaction Analysis
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The cell lysates were loaded on 10% SDS gels and blotted onto a PVDF membrane (Amersham). Santa Cruz Antibodies: anti YAP1 H-125 (1:200), anti GAPDH FL-335 (1:2000), anti p53 Do-I (1:2000) and anti ANKRD1 H-120 (1:200). Anti mp53 1C12 (1:1000) from Cell Signaling, anti MDM2 Ab-2 (1:1000) from Calbiochem. Anti BAX 06-499 (1:1000) from Millipore and anti GFP rabbit polyclonal serum (1:2000). The protein levels were quantified using ImageJ after subtraction of the background and normalized to GAPDH levels and control lysates.
For the immunoprecipitation, HEK293T cells were transiently transfected either with 10 µg of pCMV-Flag-ANKRD1 or pEYFP-ANKRD1 and empty vector control, respectively. The cell lysates were incubated overnight with anti-flag M2 agarose beads (Sigma) or with anti-GFP agarose beads (Chromotek). The beads were washed twice with cold TBS before denaturation. The cell lysates (input) and IPs were separate by SDS-PAGE. The interactions were further analyzed by western blotting using the specific antibodies.
For the immunoprecipitation, HEK293T cells were transiently transfected either with 10 µg of pCMV-Flag-ANKRD1 or pEYFP-ANKRD1 and empty vector control, respectively. The cell lysates were incubated overnight with anti-flag M2 agarose beads (Sigma) or with anti-GFP agarose beads (Chromotek). The beads were washed twice with cold TBS before denaturation. The cell lysates (input) and IPs were separate by SDS-PAGE. The interactions were further analyzed by western blotting using the specific antibodies.
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