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. 2014 Jul 10;15:28. doi: 10.1186/1471-2121-15-28

Figure 1.

Figure 1

Tandem affinity purification of 3XFLAG-TEV-CBP-Cul3 complexes. A. Diagram for the tandem affinity purification of the Cul3 binding proteins. 3XFLAG-TEV-CBP-Cul3 was first affinity purified using anti-FLAG M2 affinity gel resin. Subsequently, the Cul3 complexes were cleaved off the gel resin by the TEV protease enzyme. The eluted CBP-Cul3 complexes were affinity purified using calmodulin-conjugated beads and the Cul3 complexes were subsequently subjected to analysis by MudPIT mass spectrometry. B. Untransfected HEK293 cells (lane 1) and HEK293 cells transfected with either FLAG-tagged Cul3 (lane 2) or FLAG-TEV-CBP-Cul3 (lane 3) were harvested and sonicated in RIPA buffer. Cell lysates were immunoblotted and probed with anti-FLAG (top panel) and anti-Cul3 antibodies (bottom panel). C. Complexes containing Cul3 were isolated in the first step of the tandem affinity purification. Cell lysates containing FLAG-TEV-CBP-Cul3 protein were subjected to immunoprecipitation using anti-FLAG beads (left lane). CBP-Cul3 was released from the beads using the TEV protease (eluted fractions, lane 1). The FLAG beads were subsequently washed with TEV protease buffer (eluted fractions, lanes 2–4) and calmodulin-binding buffer (eluted fractions, lanes 5–6). The collected supernatants were immunoblotted and probed with anti-Cul3 antibody (top panel) and anti-FLAG antibody (bottom panel). D. The CBP-Cul3 complex was isolated in the second step of the tandem affinity purification. Ten milliliters of combined CBP-Cul3 fractions from the first step of the tandem affinity purification were incubated with calmodulin-conjugated beads, followed by several washes with the calmodulin-binding buffer and the calmodulin-rinsing buffer. A sample of protein-bound calmodulin beads was collected (left lane). Cul3 complexes were eluted from the calmodulin beads using the calmodulin-eluting buffer containing EGTA (lane 1–6). The eluted fractions were immunoblotted and probed with anti-Cul3 antibody. The fractions that contained the most Cul3 were selected for MudPIT analysis. See Additional file 3: Table S3.