Western Blot Visualization

Mas Rizky A.A Syamsunarno, MD, PhD

Department of Biochemistry and Molecular
Biology
Faculty of Medicine Universitas Padjadjaran
 Basic Principles

Chemiluminescence
The light signal can be captured on X-ray film or by a charge-coupled device (CCD) imager
Chemiluminescence uses enzyme-conjugated antibodies to activate the light signal
The advantages : speed and sensitivity 5 sec to 5 min
Two Systems: CDP-Star substrate (activated by Alkaline Phosphatase) or luminol (activated
by Horse Radish Peroxidase)
Detection of protein of interest

After secondary antibody incubation wash with

TBST as before.
Add enhance chemiluminance reagents
(commercially available) that have substrates for
HRP that gives a product that gives off light
energy. Capture the chemiluminescence liberated
on an X-ray film
After developing the x-ray film bands will appear.
Chemiluminescence Enhancer
troubleshooting
Example