Western Blotting

1. Cut nitrocellulose filter to pad size, mark orientation. Separate glass plates and cut off stacking gel. Moisten and assemble sandwich components (avoid air bubbles!) in transfer buffer using pyrex tray.
2. Electrophoretically transfer the proteins from the gel to the filter at 100 V constant voltage for 1.5 hours. Use cold transfer buffer, place ice tray in tank, stir. When transfer is finished, gently remove residual acryamide off the nitrocellulose filter using kimwipes.
3. Cut off protein marker lanes. Stain with amido black (0.1 % amido black, 40 % methanol, 10 % acidic acid) for 10 seconds, recycle stain. Destain on shaker with 40 % methanol, 10 % acidic acid until bands become visible. A kimwipe in the tray helps to bind excessive stain.
4. Submerge the remaning filter in blocking solution (5 % nonfat dry milk / TBS) with the "protein side up" at 4°C for overnight or 1 hour at room temperature. Cover with food wrap.
   • Weigh 2.5g nonfat dry milk into 50ml Falcon tube, add 5ml 10 X TBS + 45ml H₂O
5. Pour off blocking solution. Incubate with diluted (1% nonfat dry milk / TBS) primary antibody with the filter for 1 hour at room temperature on shaker.
   • Weigh 0.5g nonfat dry milk into 50ml Falcon tube, add 5ml 10 X TBS + 45ml H₂O + 50µl primary antibody (e.g. anti PLP-A Ab #901 in 1:1000 dilution)
6. Wash the filters three times 7 minutes with TBST (TBS / 0.05% Tween-20).
7. Incubate filter with diluted (1% nonfat dry milk / TBS) secondary antibody for 1 hour at room temperature on shaker..
   • Weigh 0.5g nonfat dry milk into 50ml Falcon tube, add 5ml 10 X TBS + 45ml H₂O + 7.2µl secondary antibody (e.g. 1:7000 goat - anti rabbit + HRP (horse radish peroxidase)).
8. Wash the filters three times 10 minutes with TBST (TBS / 0.05% Tween-20).
9. Detection using ECL (enhanced chemoluminescense) system. Mix equal volume of detection solution 1 and detection solution 2 (e.g. 1ml of each in 15ml Falcon tube), sufficient to cover membrane. Drain the excess buffer from the washed membrane by placing it on a piece of SaranWrap, protein side up. Add the detection reagent to the protein side of the membrane, so that the reagents are held by surface tension on the surface of the membrane. Do not allow the surface of the membrane to become uncovered. Incubate for precisely 1min. at room temperature without agitation. Drain off excess detection reagent by holding the membrane vertically and touching the edge of the membrane against tissue paper.
10. Wrap membrane in food wrap. Place the membrane protein side up and autoradiography film in the film cassette. Expose for 1 minute; weaker signals might require e.g. 10 minutes initial exposure. The ECL signal is completely gone after 1 hour. Remove film, immediately replace with a fresh piece of unexposed film. Do not move film while it is being exposed.

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