A question involving immunoprecipitation to identify interacting proteins?

Question

Using recombinant Flag-tagged Dcr-2 and His-tagged protein X, pull-down assays were performed to determine whether protein X and Dcr-2 interact directly. The recombinant proteins (either alone or in combination) in binding buffer (150 mM NaCl, 1 mM EDTA, 50 mM Tris pH 7.5, 1% Triton X-100) were pulled-down by Flag antibodies coupled to Sepharose beads. Following extensive washing, the proteins were eluted from the beads with SDS buffer, and duplicate sets of samples were analyzed by SDS PAGE. Samples were also taken of the proteins before they were used in the pull-down assay and these were run on the same gel (input lanes in Figure 2B). One gel was stained with Coomassie blue dye (Figure 2A), and the other was transferred to nitrocellulose for Western Blotting. The membrane was probed with a His-antibody (Figure 2B).

The question asks: Do Dcr‐2 and protein X interact directly?

My reasoning is that they DO interact directly. In Fig 2B: the intense bands on LHS shows that Dcr-2 interacts with protein X-that is bound to anti-His. In Fig 2A: the protein X interacts with the Dcr-2 bound to antibody FLAG. I feel I'm not explaining this very though...Could someone please explain their reasoning? Thank you

Answer 1

First: You are right with your reasoning that Protein X (X) and Dcr-2 interact directly. Lets go through the blots: In the input you check for the proteins loaded into the setting and stain unspecifically with Coomassie. This part of the blot shows you no unspecific input, and both proteins of interest present. This also shows that Dcr-2 is much bigger than X which allows an easy identification of band patterns.

In the eluate you will only see proteins which are bound directly in the reaction or which interact with bound proteins. Dcr-2 is Flag-tagged so it will directly bind to the beads and can be eluated from them. This is shown in the coomassie part of the blot. Dcr-2 alone binds and can be eluated, while X alone does not bind and therefore gives no signal. Dcr-2 and X together give two bands, sind Dcr-2 binds to the beads and X is interacting with Dcr-2.

This binding is also shown with the anti-His antibody which only recognizes Protein X. The protein shows up in the input where X is added. It also shows up in the elution lane (where Dcr-2 is also present) showing that X is an interaction partner for Dcr-2.