Western Blot Analysis of Epitoped-tagged Proteins Using The Chemifluorescent Detection Method - for alkaline phosphatase conjugated antibodies
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- 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:
| 1X Transfer buffer: 25 mM Tris, 192 mM Glycine, pH 8.3. Mix 3.03 g Tris and 14.4 g glycine; add water to 1 liter - do not add acid or base to pH - it should be >8.0. Use 0.5X for transfer in 20% methanol. | |
| 10X Western Buffer: 200 mM Tris pH = 7.5; 1.5 M NaCl (containing 0.1% Tween-20 and 0.2% I-Block). To prepare 1X Western Buffer, dilute 10X buffer to 1X, adding Tween-20 to 0.1%. Remove 50 ml and set aside for the last two washes. To the remainder, add I-Block to 0.2% (Cat #T2015, Applied Biosystems - formerly Tropix). To dissolve I-Block, heat solution in a beaker briefly in a microwave to about 60°C, then stir until dissolved (solution will be cloudy). Bring to room temperature before using. | |
| Primary antibody: For his tagged proteins - Anti-His monoclonal antibody - BD Bioscience #631212 | |
| Secondary antibody: Goat anti-mouse alkaline phosphatase conjugated - Biorad #170-6520. | |
| Substrate: ECF chemifluorescent substrate - Amersham #RPN5785. Mix substrate with accompanying buffer as per manufacturer’s recommended instructions, prepare 1 ml aliquots and store at -20°C. |
