1. antigen protein sample 0.1-100 µg/ml
2. blocking buffer 10% BSA in PBS 
3. primary antibody 1-2 µg/ml
4. HRP conjugated secondary antibody 1-2 µg/ml
5. rinsing buffer 1% BSA in PBS with 0.5% Tween
6. Enhanced chemiluminescent (ECL) HRP substrate

Spot 1-2 µl of antigen onto the membrane, let air dry

Spot at the center of the reaction zone.

You can prepare reagent solutions during the waiting.

Block with 50 µl blocking buffer

Inject at the center of the reaction zone. Pipette tip should be close to the membrane but do not touch the membrane.

Make sure all solutions get absorbed completely. If there is a bubble trapped at the reaction zone, use a 20 µl pipette to suck it out. 

Do not wait for more than 40 min, otherwise the membrane will get dried. 

Add 2 µl primary antibody on the membrane

Inject at the center of the reaction zone. Pipette tip should be close to the membrane but do not touch the membrane.

For higher sensitivity, you can add 20 µl of rinsing buffer onto the membrane before adding the primary antibody to reduce background. 

Add 50 µl rinsing buffer

When injecting, rest the pipette tip against the wall near the reaction zone. Do not pipette over the reaction zone as this is easy to introduce bubble. 

Make sure all solutions get absorbed completely. If there is a bubble trapped at the reaction zone, use a 20 µl pipette to suck it out. 

You can proceed to the next step as soon as all the solutions are absorbed.

Add 2 µl HRP-conjugated secondary antibody

Rinse with 50 µl rinsing buffer for 4 times

When injecting, rest the pipette tip against the wall near the reaction zone. Do not pipette over the reaction zone as this is easy to introduce bubble. 

Make sure all solutions get absorbed completely. If there is a bubble trapped at the reaction zone, use a 20 µl pipette to suck it out. 

Optional: you can rinse with 40 µl rinsing buffer for 5 times. If using a robot, try continuous dripping a total of 200 µl rinsing buffer.  

 Add 10 µl HRP substrate and detect!

Inject at the center of the reaction zone. Pipette tip should be close to the membrane but do not touch the membrane.

Measure chemiluminescent signal in a plate reader or gel imager immediately after adding the HRP substrate. Depending on the specific substrate, signal may be measured within 0-10 min.