western blot protocols, troubleshooting, scientific resources and research atricles for the research community and science students

Used daily in medicine, biology and other disciplines, the western blot method allows for the study of protein expression. The western blot protocol below should be optimized for specific experiments because of differences in antibody specificity, protein copy number and other parameters.

1. PubMed: comprehensive database - 15 million citations

2. NIH:the National Institutes of Health

3. UniProt: The world's most comprehensive catalogue of information on proteins

4. Western Blot Procedure Western blot protocol overview.

After completing SDS-PAGE and electrophoretic transfer, block the membrane in 5% Non Fat Dry Milk (NFDM) in TBS with 0.05% tween-20 for 2 hours. A good way to accomplish this is by using 50 ml of blocking solution for 1 small membrane: pour the solution into a 50 ml centrifuge tube, insert the membrane (protein side facing towards the inside of the tube), close the tube and place on a rotating hematology mixer to rotate for 2 hours.

Incubate the membrane with the primary antibody for 16 hours at 4 degrees C. Dilute the antibody with a 2.5% Non Fat Dry Milk (NFDM) in Tween TBS solution. A total antibody and diluent solution of 5 ml will coat a small membrane in a rotating tube very well.

Remove the antibody and perform 3 room temperature washes (10 minutes of rotation each) with Tween TBS.

Reblock the membrane in 10% Non Fat Dry Milk (NFDM) in Tween TBS for 10 minutes at room temperature.

Incubate the membrane with the secondary antibody for 30 minutes at room temperature. Dilute the antibody with a 2.5% Non Fat Dry Milk (NFDM) in Tween TBS solution. You can also add blocking serum from the same species in which the secondary antibody was produced.

Remove the antibody and perform 3 room temperature washes (10 minutes of rotation each) with Tween TBS.

Prepare the chemiluminscent reagents. It is important that you prepare the ECL solution just prior to use in order to maximize its effectiveness.

Lay the membrane face up on an acetate

Pour the chemiluminscent solution over the membrane, covering it completely.

After 30 seconds to incubate, place a second acetate over the top and squeeze away excess solution.

Turn out the lights and place the membrane/acetate sandwich in a film cassette with the appropriate film. Exposure times are extremely variable and some care should be taken to determine the optimal exposure parameters.

Develop film; remember to use a fixing solution.