1. Heat samples in Laemmli buffer at 70°C for 10 min.
2. Load 80-100 µg/lane of membranes/lysates sample or 2-5×105 cell lysate/lane.
3. Load samples on gel. For high molecular weight proteins Tris-Glycine 4-6% or Tris-Acetate 3-8% are the best options and should be chosen experimentally.
100 mA for 22h at 4°C.
Note: This step is crucial since it determines the effectiveness of the transfer. A longer transfer time will increase the efficiency of the transfer of proteins of high molecular weight.
1. Block membranes with blocking solution (3% BSA in PBS, 0.05% NaN3) for 2-5 hr at room temperature with gentle agitation.
2. Add primary antibody diluted in PBS with 1% BSA, 0.1% Tween-20 and 0.05% NaN3 at the appropriate dilution. Incubate overnight at 4°C with gentle agitation. (If negative control antigen is used, refer to Negative Control Antigen Manipulation protocol).
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 on if you are using a secondary antibody conjugated to horseradish peroxidase (HRP).
4. Incubate secondary antibody at appropriate dilution in PBS with 1% BSA, 0.1% Tween-20 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.
7. Exposure time to film/imager depends on the abundance of the protein and the detection system.
Buffers & Solutions
Primary antibody solution:
Secondary antibody solution: