Protein detection by western blot
The molecules that have been separated are moved or "blotted" onto a second matrix, which is usually made of nitrocellulose or polyvinylidene difluoride. The membrane is blocked so that antibodies can't bind to its surface in a way that isn't what they are supposed to do. Often, the secondary antibody is bound to an enzyme, which, when mixed with the right substrate, makes a signal that can be picked up.
How to Make a Good Western Blot
The standard way to see the target protein is to let the primary antibody mix with the sample for one hour at room temperature. But an overnight incubation at 4°C should give the antibody-antigen reaction plenty of time, making it more likely that a positive signal will be shown. It also help to perform all washing steps at 4°C, that can be done using BlotCycle, automated western blot processor
Primary antibody incubation time
During the detection step, the complex of the protein, antibody, and antibody is found on the membrane. Detection systems can be based on chemiluminescence, chemifluorescence, fluorescence, chromogenic, or radioisotopic detection. You can use a film, a CCD camera, or a scanner to collect the light given off by the detection process during the capturing step. Most of the time, it's easy to figure out what the data from a Western blot means.