Now you transfect these cells  (which already contain the tetracyclin transactivator) with a plasmid containing the BHR1 gene under control of the tetracyclin controlled promoter. The plasmid you use confers resistance to hygromycin.  See the figure to the right.

Following transfection you start a seven days selection period in the presence of  G-418 + hygromycin. At the end of this period you have to determine which clones incorporated the BHR1 gene into their genome and now express it on their plasma membrane. You will do that in two different ways:

1. Lyse the cells, run the cell lysates in PAGE-SDS gels and do Western Blots using an anti BHR1 antibody.
2. Use an anti BHR1 antibody to detect the receptors on the cell surface of intact cells.

Four clones were grown in the absence of tetracyclin(inducing expression). Cells were lysed and samples were loaded on a gel for a western blot. () Here are the results:

You now proceed with the second assay - using an anti BHR1 antibody to detect the receptors in intact cells.
Each of the same four cell-lines was plated into two different dishes. A day later you added tet to one well from each cell line and added only the tet buffer (blank reagents) to the other well. After two more days you  exposed the cells to mouse anti BHR1 antibody (=primary antibody) and then to goat anti mouse antibody (=secondary antibody) labeled with rodamine (red fluoresence) and examined the cells in a confocal microscope. The results are presented below (Click on any clone number to get more details).
 

Clone number + TETRACYCLIN (Repressed expression) - TETRACYCLIN (Induced expression) Noam-10-1 Noam-10-2 Noam-10-3 Noam-10-4

This is a reminder of the results obtained by Western blot analysis. The following conclusions are based on results obtained using the two procedures.  Choose between right and wrong. 1. In Noam-10-1 clone the expression is leaky, and that is why there is no difference between induced and non-induced cells. 2. In Noam-10-1 cells there is no difference between induced and non-induced cells due to some technical problem during the reactions with the primary and secondary antibodies. 3. Noam-10-2 cells express high amounts of BHR1 in the plasma membrane. 4.  Noam-10-2 cells express high amounts of BHR1, but we don't know where the protein is found in the cell. 5. Noam-10-3 cells express lower amounts of BHR1 in the plasma membrane, because the overall expression of BHR1 in these cells is lower. 6. Noam-10-4 cells don't express any BHR1 protein whithin the cells. 7. Noam-10-4 cells don't express any BHR1 protein in the plasma membrane. BHR1 is found in another location whithin the cell.