Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies
- 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 the 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 for 1 hr at 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 at room temperature, gently rocking. Wash 3 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 for 30 min at room temperature.
- Wash as in step 5. Additionally, 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 pipette (with a p10000) the solution over the membrane to ensure the entire surface comes into contact with the substrate. Gently agitate for a few minutes, remove the membrane to a paper towel, and let it dry completely. The substrate solution can be reused immediately for additional membranes.
- Scan membrane using the molecular dynamics storm.
Western Blotting Solutions
- 10X Transfer buffer: 240 mM Tris, pH = 8.0; 1.92 M Glycine. Use 0.5X for
transfer in 20% methanol. - 10X Western buffer: 200 mM Tris pH = 7.5; 1.5 M NaCl. 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 2 washes. To the remainder, add I-Block to 0.2%, heating gently with constant stirring until dissolved. Bring to room temperature before using (containing 0.1% Tween-20 and 0.2% I-Block).
- Primary antibody: For his tagged proteins - Anti-His monoclonal antibody.
- Secondary antibody: Goat anti-mouse alkaline phosphatase conjugated - Biorad #170-6520.
- Substrate: ECF chemifluorescent substrate—Mix substrate with accompanying buffer as per manufacturer’s recommended instructions, prepare 1-mL aliquots, and store at –20°;C.