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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.
  2. 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.
  3. 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.
  4. Block in 30 ml of 1X Western buffer (containing 0.1% Tween-20 and 0.2% I-Block), gently rocking, 1 hr, room temperature.
  5. 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.
  6. Wash three times in 20 ml 1X Western buffer (containing 0.1% Tween-20 and 0.2% I-Block) for 5 min each.
  7. 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.
  8. Wash as in step #6.
  9. Then, wash twice with 1X Western buffer without I-block.
  10. 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.
  11. Scan membrane using the Molecular Dynamics Storm or other suitable instrument.

Western Blotting Solutions:

bullet 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.
bullet 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.
bullet Primary antibody: For his tagged proteins - Anti-His monoclonal antibody - BD Bioscience #631212
bullet Secondary antibody: Goat anti-mouse alkaline phosphatase conjugated - Biorad #170-6520.
bullet 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.

This Web page is maintained by Julie B. Wolf, UMBC;
Last updated on 3/2/2010

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