TT-Ago2 (control), TT-FHAgo1, or TT-FHAgo2 cells were induced with Dox (2.5 µg/mL) and fractionated to cytosolic and nuclear fractions. Cytosolic and nuclear fractions were dialyzed in buffer DB (20 mM Tris-Cl pH 8, 10% Glycerol, 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA) for 6 h with four buffer changes. Dialyzed lysates were centrifuged and Flag IP was carried out overnight with M2 Flag conjugated magnetic beads (Sigma). Immunoprecipitated material was washed and eluted with 3× Flag peptide (Sigma) twice at room temperature. The eluted material was TCA precipitated overnight at 4°C. The precipitate was resuspended in gel loading buffer, separated utilizing 4%–12% SDS-PAGE (Thermo Fisher Scientific), and visualized with Imperial stain (Thermo Fisher Scientific). The gel was separated into eight equal size pieces and submitted to the Biopolymers & Proteomics Core Facility of Robert A. Swanson (1969) Biotechnology Center at the Koch Institute for mass spectrometry.
M2 flag conjugated magnetic beads
Manufactured by Merck Group
The M2 Flag conjugated magnetic beads are a product manufactured by Merck Group. They are composed of magnetic particles that have been conjugated with the M2 Flag peptide sequence. The core function of these beads is to provide a means for the capture, isolation, and purification of proteins or other biomolecules that contain the M2 Flag tag.
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Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (2 mentions)
Imperial stain, by Thermo Fisher Scientific (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
Flag peptide, by Merck Group (1 mentions)
Poly d lysine coated plates, by Merck Group (1 mentions)
Actn05, by Abcam (1 mentions)
Anti mouse nxa931, by GE Healthcare (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
2 protocols using m2 flag conjugated magnetic beads
1
Identification of Ago2-Associated Proteins
2
PTRN-1 Protein Detection and Co-Immunoprecipitation
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Worm protein samples were prepared by boiling 10 μl pellets of mixed-stage worms in SDS-mercaptoethanol solution. We detected PTRN-1 using antibody OD208A (Wang et al., 2015 (link)); we used the anti-actin antibody ACTN05 (Abcam, Cambridge, MA, C4) as a loading control. We used HRP-linked anti-rabbit IgG NA934V and anti-mouse NXA931 as secondary antibodies (GE Healthcare Lifesciences) at 1:1000 dilution in TBS, and added SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA). The blot result was replicated once.
To test co-immunoprecipitation, a 1:1 ratio of tagged DAPK-1 and PTRN-1 were co-transfected using Lipofectamine 2000 (Invitrogen) into HEK293 cells growing on poly-D-Lysine-coated plates (Sigma-Aldrich) in Opti-MEM (Thermo Fisher Scientific). After two days, cells were collected in cold PBS and lysed in lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% NP40, 0.5 mM EDTA, 3 mM MgCl2) for 30 min at 4°C. M2-FLAG conjugated magnetic beads (Sigma) were used for IP, and mouse anti FLAG (F1807, Sigma, 1:1000) and anti HA (HA-7, Sigma, 1:5000) antibodies were used for western blotting. The co-IP was repeated twice.
To test co-immunoprecipitation, a 1:1 ratio of tagged DAPK-1 and PTRN-1 were co-transfected using Lipofectamine 2000 (Invitrogen) into HEK293 cells growing on poly-D-Lysine-coated plates (Sigma-Aldrich) in Opti-MEM (Thermo Fisher Scientific). After two days, cells were collected in cold PBS and lysed in lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% NP40, 0.5 mM EDTA, 3 mM MgCl2) for 30 min at 4°C. M2-FLAG conjugated magnetic beads (Sigma) were used for IP, and mouse anti FLAG (F1807, Sigma, 1:1000) and anti HA (HA-7, Sigma, 1:5000) antibodies were used for western blotting. The co-IP was repeated twice.
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