western blots of miniTurboID- or degron-tagged mESCs

Description

Western blot analysis of mouse embryonic cells expressing endogenously p300-miniTurboID-V5-T2A-puromycin, PAXIP1-miniTurboID, SP5-HA-FKBP12F36V, RXRalpha-FKBP12F36V or ZEB2-HA-FKBP12F36V.

Steps to reproduce

HA pulldown followed by western blotting Approximately 500μg of protein lysate per reaction was incubated with 15μl prewashed anti-HA agarose beads (Sigma, A2095) and 2μl of ethidium bromide (Sigma, 10mg/ml) for 90 min in a rotation wheel at 4 °C. The beads were washed twice with RIPA buffer, twice with PBS + 1% NP40 and twice with PBS. Beads were resuspended in Laemmli buffer (120mM Tris, 20% Glycerol, 4% SDS, 100mM DTT, CPI and bromophenol blue) and boiled for 10min at 95°C after which proteins were separated on an SDS–PAGE gel. Proteins were transferred from the gel to a nitrocellulose membrane using wet transfer. Nitrocellulose membranes were blocked with 5% milk in 0.1% Tween-PBS for 30 min followed by 1h primary antibody (anti-HA clone 3f10, Roche, 11867423001) and 1h secondary antibody (anti-rat Ig/HRP, Agilent Dako, P0450). Streptavidin pulldowns Cells were treated with 50μM biotin (B20656, Life technologies) for 1 hour at 37°C. Cells were harvested by scraping and gastruloids by centrifugation. Pelleted cells were washed twice with PBS and resuspended in five volumes of RIPA buffer (150mM NaCl, 50mM Tris pH 8.0, 1mM EDTA, 10% glycerol, 1% NP40, 1mM DTT and CPI). Lysates were rotated for 1 hour at 4°C and centrifuged for 5 min at 21,000g at 4°C. The supernatant was collected and protein concentration was measured using BCA protein assay (Thermo FIsher Scientific). Approximately 500μg of protein per reaction was incubated with 15μl prewashed Streptavidin Sepharose High-Performance beads (15511301, Cytiva) and 2μl of ethidium bromide (Sigma, E1510) for 2 hours in a rotation wheel at 4 °C. The beads were washed twice with RIPA buffer, twice with PBS + 1% NP40 and twice with PBS. Beads were resuspended in Laemmli buffer (120mM Tris, 20% Glycerol, 4% SDS, 100mM DTT and CPI) and boiled for 10min at 95°C for western blot. 20ug of protein was loaded as input.

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