1. Heat samples in Laemmli buffer at 70°C – 90°C for 10 min.
2. Load 80-100 μg/lane of membranes/lysates sample or 2-5×105cell lysate/lane.
3. Perform SDS-PAGE according to standard protocols.
Perform transfer according to the manufacturer’s instructions.
Note: High molecular weight proteins may need longer transfer times.
1. Block membranes with blocking solution (3% BSA in PBS + 0.1% Tween-20 and 0.05% NaN3) for 2-5 hr at room temperature with gentle agitation.
2. Add primary antibody diluted in blocking solution at the appropriate dilution. Incubate for 2-3 hr at room temperature or overnight at 4°C with gentle agitation. (If control antigen is used, refer to the protocol describing the use of control antigen).
3. Discard primary antibody and wash membranes by incubating with washing buffer (PBS + 0.1% Tween-20) 3 times for 15 min at room temperature.
Note: Do not use solutions containing NaN3 from this point if you are using a secondary antibody conjugated to horseradish peroxidase (HRP).
4. Incubate secondary antibody at appropriate dilution in washing buffer for 1 hr at room temperature with gentle agitation.
5. Wash membranes with washing buffer 3 times for 15 min at room temperature.
6. Proceed to detection using an ECL system. If using a commercial kit, perform according to the manufacturer’s instructions.