CHO-S cells were transfected with C-terminally HA-tagged EGFR, and stable colonies were selected. Cells were stimulated with 100 ng/mL EGF for 5 min. in 24-well plates, then lysed with RIPA buffer. Unstimulated control wells were also lysed in the same way. Anti-HA antibody was added to the lysates and incubated overnight with Protein G beads. The beads were then washed, eluted with SDS-PAGE loading buffer, and precipitated protein run on SDS-PAGE gels for Western blotting.
The arrow is at the expected position of EGFR. The doublet between 20-30 kDa is due to Protein G, which is also eluted from the resin and retains some antibody binding ability. The protein G band binds primary/secondary Western antibodies and is therefore detected.
Note that the given antibody dilutions are appropriate only for Westerns using the SNAP i.d. device (Millipore). For a standard western, multiply the dilution by 3/10.
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