Lab Experiment 15.1: Analyze the E.Coli Over-expressing cells for the expression of GFP inside the cells.

Background Information: The western blot is a popular technique to detect the specific protein present in the crude lysate or homogenate. It uses the separation of different proteins in the gel electrophoresis (preferably SDS-PAGE) and then the transfer of protein onto a solid support such as nitrocellulose membrane. A primary antibody direct against protein of interest and then a secondary antibody is used to detect the primary antibody and gives either colored or chemiluminescent signal.

Figure 15.1: Salient Steps in Western Blotting.

Materials:

1. E.coli cells over-expressing GFP.

2. Protein Standard Markers

3. Transfer Buffer

4. Transfer Membrane: 0.45µm nitrocellulose membrane or PVDF membrane.

5. Plastic Tray

6. Spatula

7. Blotting sheets: 3mm thick cellulose blotting sheets.

8. Semi-Dry Electroblotting Unit.

9. Reagents for performing electrophoresis.

10. anti-GFP antibodies

11. anti rabbit IgG-HRP.

12. Developing reagents