Summary: Western ECL Detection
· Trim blot with scissors to minimize area. Cut notch at bottom right corner for orientation. Clean
plastic trays to reduce background and improve sensitivity. Do all steps at room temperature.
1. Prepare PBSTwM blocking solution: Dissolve 2.5 g non-fat dry milk in 45 ml dH2O. Stir for ~15
minutes. Add 5 ml 10X PBS. Add 0.25 ml 10% (wt/vol) Tween-20.
2. Blocking: Transfer blot to PBSTwM solution. If blot is dry, float on surface of blocking solution
to allow slow and even wetting. Block for 30 minutes with gentle agitation.
3. Rinse: Rinse membrane with PBSTw: 2 X 1 minute
1 X 15 minutes
2 X 5 minutes
PBSTw 1X PBS + 0.05% (wt/vol) Tween 20
4. Primary antibody: Add 1° ab diluted 1/10,000 in PBSTw (no milk). Gently agitate for 1-2 hours.
Optimal antibody concentration is determined empirically. If unsure, try 1/2,000 - 1/20,000. Some
antibodies work better if incubated at 4°C (2 hours to overnight). If diluted antibody will be
reused, add 1 mM EDTA and store at 4°C.
5. Rinse membrane with PBSTw as described in step 3.
6. Secondary antibody: Add 2° HRP-anti-Mo or HRP-anti-Rb ab diluted 1/10,000 in PBSTw.
Agitate 1 hour. Do not reuse 2° antibody. Never add sodium azide to HRP-conjugated antibodies.
7. Rinse membrane with PBSTw as described in step 3.
8. ECL set up: While rinsing in step 7, set up for ECL detection in dark room.