Protein detection: Specific antibody-mediated detection of proteins on a membrane
Working with antibodies
Detection of a protein on a membrane
Immunodetection of a protein immobilized on a membrane
Immunodetection using a chromogenic method
Perform all incubation and wash steps on a rocking platform or orbital shaker.
- Follow steps 1–7 of the protocol Immunodetection using a chemiluminescent method
- Incubate the membrane with secondary antibody solution diluted in 3% BSA (w/v) in TBS for 1 h at room temperature. Dilute according to the manufacturer’s recommendations.
Tip: Ensure that your secondary antibody is directed against the species of origin of your primary antibody.
Tip: Use the lowest recommended concentration to avoid false signals.
- Wash 4 times for 10 min each time in TBS-Tween/Triton buffer at room temperature.
- Stain with AP or HRP staining solution until the signal is clearly visible (approximately 5–15 min). Do not shake blots during color development.
Tip: Ensure that you use the correct chromogenic detection substrate, i.e., an AP substrate for AP conjugates, or an HRP substrate for HRP conjugates.
- Stop the chromogenic reaction by rinsing the membrane twice with water.
- Dry the membrane and photograph as soon as possible as the colors will fade with time. The product formed when using HRP is particularly unstable.