|
| Step: |
Electrophoresis and blotting |
Action: |
According to the usual protocols |
Remarks: |
Use Nitrocellulose or PVDF membrane |
|
| Step: |
Membrane Blocking |
Action: |
Block membrane with blocking solution, TBS-T or PBS-T with 5% dried milk (w/v), under constant shaking |
Volume: |
0.5ml/cm2 |
Time: |
1 hour at room temperature |
Remarks: |
Alternatively: overnight at 4ºC without shaking |
|
| Step: |
Primary Antibody |
Action: |
Dilute the primary antibody in TBS-T or PBS-T with 2% dried milk (w/v). Incubate the membrane in solution with shaking. |
Volume: |
0.1ml/ cm2 |
Time: |
1 hour at room temperature |
Remarks: |
Alternatively: overnight at 4ºC without shaking |
|
| Step: |
Washing |
Action: |
Three times with TBS-T under constant shaking |
Volume: |
0.5ml/ cm2 |
Time: |
3 x 10 minutes |
|
| Step: |
Secondary Antibody |
Action: |
Dilute the HRP-labeled secondary antibody (1:10,000-1:60,000) in TBS-T or PBS-T with 2% dried milk (w/v). incubate the membrane in the solution |
Volume: |
0.1ml/ cm2 |
Time: |
1 hour at room temperature |
|
| Step: |
Washing |
Action: |
Three times with TBS-T under constant shaking |
Volume: |
0.5ml/ cm2 |
Time: |
3 x 10 minutes |
|
| Step: |
Equilibration |
Action: |
Mix equal volumes of EZ-ECL Solution A&B |
Volume: |
0.1ml/ cm2 |
Time: |
5 minutes |
|
| Step: |
Detection |
Action: |
Incubate membrane in detection solution |
Volume: |
0.1ml/ cm2 |
Time: |
1-3 minutes |
Remarks: |
With gentle shaking |
|
| Step: |
Exposure |
Action: |
Remove excess detection mix, wrap in saran wrap and expose to film |
Time: |
0.5-60 minutes |
Remarks: |
Remove air pockets |
|
| |
