Western Blot Analysis of Epitoped-tagged Proteins Using The Chemifluorescent Detection Method
- Cut PVDF membrane to the appropriate size, activate with absolute methanol for 5 sec, and incubate in distilled water for 5 min.
- For electroblotting, equilibrate in transfer buffer and follow the standard blotting procedure to transfer the proteins to the membrane. For dot blotting, keep membrane wet until ready to use.
- After protein has been transferred to the membrane, wash again in absolute methanol for a few seconds and allow to dry at room temperature for 30 min. or more.
- Block in 30 ml of 1X Western buffer (containing 0.1% Tween-20 and 0.2% I-Block), gently rocking, 1 hr, room temperature.
- Add appropriate dilution of primary antibody (typically 1:5000 or 1:10,000) prepared in 1X Western buffer (containing 0.1% Tween-20 and 0.2% I-Block), incubate 30 min, room temperature, gently rocking.
- Wash three times in 20 ml 1X Western buffer (containing 0.1% Tween-20 and 0.2% I-Block) for 5 min each. Add appropriate dilution of secondary antibody conjugated to alkaline phosphatase prepared in 1X Western buffer (containing 0.1% Tween-20 and 0.2% I-Block), gently rocking, 30 min, room temperature.
- Wash as in step #6.
- Then, wash twice with 1X Western buffer without I-block.
- At the end of the second final wash, leave some buffer in the container to keep the membrane moist. With the membrane facing protein-side up, add 0.5 ml of substrate solution directly into the remaining liquid, mix well, and pipet (with a p1000) the solution over the membrane to ensure the entire surface comes into contact with the substrate. Gently agitate for a few minutes, remove membrane to a paper towel and let dry completely. The substrate solution can be reused immediately for additional membranes.
- Scan membrane using the Molecular Dynamics Storm or other suitable instrument.
Western Blotting Solutions: