Western Immunoblotting of Proteins

1. Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a cell sample prepared using modified and transfer the proteins to a nitrocellulose membrane.

2. Wash the nitrocellulose twice with distilled ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA">H₂O. Stain the nitrocellulose with Ponceau Red solution for 5 minutes to visual protein bands (Stock solution: 2% Ponceau S in 30% trichloroacetic acid and 30% sulfosalicylic acid; dilute 1:10 for use). Rinse the membrane in H₂O until protein bands are distinct and mark the position of the molecular weight markers with a ball point pen or pencil. The Ponceau Red stain will be washed off the membrane during the blocking step.

[Note: Do not let the blot dry out at any step through development as this will cause an increase in background staining.]

3. Block the blotted nitrocellulose membrane in freshly prepared PBS containing 3% nonfat dry milk for 20 minutes at room temperature with constant agitation. A maximum blocking time of 2 hours at room temperature should not be exceeded since staining artifact may appear. For longer blocking times, the membrane should be kept at 4℃.

4. Dilute the primary antibody to the recommended concentration/dilution in fresh PBS/3% nonfat dry milk (please see Certificate of Analysis for detailed information). Place the nitrocellulose membrane in the primary antibody solution and incubate 1 to 2 hours at room temperature or overnight at 4℃ with agitation.

5. Wash the nitrocellulose membrane five times for 3 to 5 minutes each with either water or PBS containing 0.05% Tween 20.

6. Incubate the nitrocellulose membrane in the secondary reagent of choice for 30 minutes to 1 hour at room temperature or overnight at 4℃ with agitation. For a mouse monoclonal antibody, a goat-anti-mouse HRP conjugated antibody is recommended, for a rabbit polyclonal antibody, a goat-anti-rabbit HRP conjugated antibody is advisable.

7. Wash the nitrocellulose membrane five times for 3 to 5 minutes each with either water or PBS containing 0.05% Tween 20. If the membrane has been washed with water, a final wash step for 3 to 5 minutes in PBS containing 0.05% Tween 20 should follow.

8. Rinse the nitrocellulose briefly in 4 to 5 changes of water, then perform detection of proteins using a detection system of choice, e.g., enhanced chemiluminescence (ECL).

TIP: If it does not inhibit antibody binding, Tween 20 (0.05% final concentration) can be added to the blocking and antibody solutions as well to reduce nonspecific background staining.