A total of 5 × 105 293T cells were seeded in each well of a 6-well plate 12 h before transfection. Forty-eight hours posttransfection, cells were lysed by RIPA buffer. Cytosolic fractions were collected and incubated with anti-FLAG antibody-conjugated magnetic beads (Sigma) overnight at 4°C. The beads were isolated by magnetic shelf, washed several times, and used for WB assay.
Anti flag antibody conjugated magnetic beads
Manufactured by Merck Group
Anti-FLAG antibody-conjugated magnetic beads are a lab equipment product designed for the isolation and purification of FLAG-tagged proteins. The beads are conjugated with anti-FLAG antibodies, allowing for the specific capture and separation of FLAG-tagged proteins from complex samples. The magnetic properties of the beads facilitate easy handling and separation using a magnetic separator.
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4 protocols using anti flag antibody conjugated magnetic beads
1
FLAG-tagged protein immunoprecipitation
2
Flag-Protein Immunoprecipitation and Analysis
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p3xFLAG-TMSB10, p3xFLAG-CMV10, and pCMV-HA-KRAS were transfected using the lipofectamine 2000 reagent (Thermo Fisher Scientific) in HEK293 cells. The cells were lysed in 50 mM Tris–HCl (pH 8.0), 300 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM EDTA, and 1% Triton X-100. The p3xFLAG-TMSB10 and p3xFLAG-CMV10 were immunoprecipitated with anti-FLAG antibody-conjugated magnetic beads (Sigma-Aldrich), and then the beads were sequentially washed three times with washing buffer (20 mM HEPES-KOH (pH 7.6), 100 mM KCl, 10% glycerol,1 mM EDTA, and 0.05% Tween20), and once with PBS. Finally, the immunoprecipitates were eluted from the beads with 10 μl elution buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 500 μg/ml FLAG peptide). Eluates and inputs were subjected to SDS–polyacrylamide gel electrophoresis followed by Western blotting. The antibodies used in this study are listed in Supplementary Table 6 .
3
Biochemical Characterization of KaiB Interactions
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Purified proteins of interest were coincubated with KaiB-FLAG or fsKaiB-FLAG in 30 °C. After the incubation, the reactions were snap-frozen in liquid nitrogen and kept in –80 °C. The frozen reactions were thawed on ice and were promptly subjected to co-IP using anti-FLAG antibody-conjugated magnetic beads (Sigma-Aldrich, #M8823). The eluates from the co-IP were run in SDS-PAGE gel. The gel was stained using Sypro Ruby (Invitrogen), which was then imaged by ChemiDoc (Bio-Rad). The band intensity was used as a metric of the quantity of each protein that was pulled down by the anti-FLAG beads (see In Vitro Co-IP Using FLAG-Tagged Proteins as Bait in SI Appendix, Supplementary Materials and Methods for details). Fluorescence polarization measurements were performed by adding fluorescein-labeled KaiB to the purified KaiABC reaction as previously described (3 (link), 28 (link), 29 ) (see Fluorescence Polarization in SI Appendix, Supplementary Materials and Methods for details).
4
Chromatin Immunoprecipitation of FLAG-tagged Proteins
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Control or Cdt2-FLAG expressing 293 cells that were UV irradiated (50 J/m2), or not UV irradiated, or in the middle S phase (5 h after release from aphidicolon arrest) were fixed with 0.02% formaldehyde for 10 min, lysed using 0.1% Triton X-100-containing modified cytoskeleton (mCSK) buffer (10 mM Pipes, pH 7.9, 100 mM NaCl, 300 mM sucrose, 0.1% [vol/vol] Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 mM β-glycerophosphate, 1 mMNa3VO4, 10 mM NaF), and then sonicated. After centrifugation (120,000g, Beckman Type 45Ti rotor, for 20 min at 4°C), the supernatants were mixed with anti-FLAG antibody-conjugated magnetic beads (M8823; Sigma-Aldrich) for 1 h at 4°C to obtain immunoprecipitates. The precipitate was washed with ice-cold 0.1% Triton X-100 containing mCSK buffer and subsequently suspended in SDS sample buffer.
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