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Home > Molecular Biology > Protein > Western Blotting

Western Blot Protocol with Anti-HA

The folllowing protocol is suitable for detecting HA-tagged histone proteins in yeast:

  1. Run the cell lysate by SDS-polyacrylamide gel electrophoresis, transfer the proteins onto nitrocellulose membrane.
  2. Wash the blotted nitrocellulose twice with ultrapure water.
  3. Block the membrane with 5% nonfat dry milk in TBS for 20 minutes.
  4. Incubate the nitrocellulose with a 1: 2,000 dilution of anti-HA-Tag, diluted in freshly prepared TBS-milk, for two hours at room temperature (Use anti-H3 at 1: 4,000 dilution).
  5. Wash twice with ultrapure water, 5 minutes each.
  6. Incubate the nitrocellulose with secondary antibody (goat anti-rabbit HRP conjugated IgG) for 1.5 hours at room temperature, diluted in TBS-MLK (1: 5,000).

    7. Note: Never use Sodium Azide (NaN3) during secondary antibody incubation. It irreversibly inhibits HRP. You will not detect any signal.

  7. Wash the nitrocellulose twice with ultrapure water, 5 minutes each.
  8. Wash the nitrocellulose once in TBS-0.05% Tween-20 for 5 minutes.
  9. Rinse the nitrocellulose in 4-5 changes of water.
  10. Use chemiluminescence for detection.

Anti-HA-tag, clone DW2, rabbit monoclonal IgG, Millipore Cat No. 05-902. Anti-Histone H3, rabbit polyclonal, Abcam Cat No. ab1791.

by Erbay Yigit