1. Separate proteins by running a PAGE gel.

2. Transfer proteins onto a Nitrocellulose or PVDF membrane.

3. Make TBS buffer: (50 mM Tris pH 8.0, 150 mM NaCl). Make a 10X TBS buffer (43.5g NaCl, 500 ml 1M Tris-HCl pH 8.0, add water to 1 liter) and dilute.

4. Briefly rinse the membrane in TBS.

5. Place the membrane in TBS/0.1%Tween-20/5% skim milk, block at 4 °C for 2 hours to overnight. Use a container about the size of the membrane or a hybridization bag.

6. Wash with TBS/0.1%Tween-20 for 2 minutes twice.

7. Dilute the primary antibody to required concentration in TBS/0.1%Tween-20/5% skim milk. Add to the membrane and hybridize for 2-4 hours at room temperature or overnight at 4 °C.

8. Wash the membrane with several changes of TBS/0.1%Tween-20 over a period of at least 30 minutes.

9. Dilute the primary antibody to required concentration in TBS/0.1%Tween-20/5% skim milk. Add to the membrane and hybridize for 1-2 hours at room temperature or overnight at 4 °C.

10. Wash the membrane with several changes of TBS/0.1%Tween-20 over a period of at least 45 minutes.

11. Transfer the membrane to a Saran wrap on a flat surface. Touch the edge of the membrane to a paper towel to drain off excess wash solution. Place the membrane protein side up.

12. Mix 1:1 ECL solution 1 and 2.

13. Add the mixed ECL solution onto the membrane. Wait for 1 minutes.

14. Transfer the membrane to a new Saran wrap, protein side down. Wrap the membrane. Make sure there is no air bubbles and the Saran wrap don't wrinkle on the protein side.

15. Expose the membrane to a X-ray film in the dark room, protein side facing the film. You may tape the membrane/Saran wrap to a card board or an intensifying screen to avoid the membrane being inserted into the X-ray film developer.

16. Test expose for 10 seconds to decide the optimal exposure time. Expose for 5 seconds to 1 hour.