Figure 2.

Tandem affinity purification. (A) A Schematic representation of the two steps of TAP-14-3-3 purification. The two-step procedure decreases the non-specific interactions. The purified proteins can be identified by protein gel blot analysis and/or mass spectrometry. (B) TAP-14-3-3 isolation was performed as described in the materials and methods. Detection of TAP-14-3-3 and endogenous 14-3-3 levels at each step of purification by protein gel blot analysis using an antibody against 14-3-3. (C) The EGTA eluate containing the 14-3-3 binding proteins was concentrated after TAP. The proteins were separated on a 12% SDS-PAGE stained with coomassie blue and the gel was used for the LC-MS/MS analysis.