Question

2. What is NOT one of the useful features of adding a myc or FLAG tag to a protein in a construct?

A You can identify exogenous proteins versus the endogenous version(s)

B It protects the protein from proteolysis

C It will allow selective recovery of the tagged protein by immunoprecipitation

D It allows use of commercially-available antibodies when there are no existing antibodies for your target protein

3. Tet-ON and Tet-OFF systems are very similar in their overall design and use similar components. However, why is Tet-ON in many cases a superior approach than Tet-OFF?

A There can be residual mRNA that remains after activating a Tet-OFF construct

B Tet-OFF systems require more serum

C Tet-OFF systems more frequently result in excess expression of target proteins

D Tet-OFF systems are less stable

Answer 1

2 D

The gene for the target protein plus a short DNA segment encoding FLAG is cloned into a vector, and the hybrid protein is produced in either bacteria or a cell line. FLAG-tagged proteins can be isolated from a cell lysate using the anti-FLAG antibody either bound to beads or attached to a column. Only the tagged protein attaches to the beads/column. Finally, the FLAG-tagged protein is separated from the antibody by adding free FLAG peptide. The short peptide is present in surplus and competes for antibody with the tagged protein, which is therefore eluted from the beads or column.

3 B

The Tet-Off system has been studied more extensively than the Tet-On system, the two systems are truly complementary. When properly optimized, both systems give tight on/off control of gene expression, regulated dose-dependent induction with similar kinetics of induction, and high absolute levels of gene expression. Thus, for most purposes, there is no inherent advantage of using one system over the other. With the Tet-Off system, it is necessary to keep Tc or Dox in the medium to maintain the native (off) state. Because Tc and Dox have relatively short half-lives (see below), you must add Tc or Dox to the medium at least every 48 hours to suppress expression of Gene X. Conversely, in the Tet-On system, the native (off) state is maintained until induction. For this reason, Tet-On may be more convenient in transgenic applications, because you need only add Dox to the animals' diet when induction is desired.