Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. Wash as in step 5. Additionally, wash twice with 1X Western buffer without I-block.
  6. 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.
  7. 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.

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