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Enhanced chemiluminescence (ECL) immunodetection procedure
We carry out the whole procedure in a rotating oven at room temperature. The use of a nucleic acid glass hybridizer tube minimizes the volumes and costs.
- Block the membrane in 10 ml of a solution containing PBS (pH 7.2) and nonfat dry milk (5% w/v) for 30 min.
- Incubate the membrane in 10 ml of a solution containing PBS-Tween 20 (0.5% v/v), nonfat dry milk (5% w/v) and the primary antibody/antibodies (1:100 or greater, depending on Ab) for 2 h.
- Perform three quick rinses with 10 ml of PBS-Tween 20 (0.5% v/v) and then wash the membrane for 3 x 10 min with 10 ml of PBS-Tween 20 (0.5% v/v).
- Incubate the membrane in 10 ml of a solution containing PBS-Tween 20 (0.5% v/v), nonfat dry milk (5% w/v) and the secondary peroxidase-conjugated antibody (1:1000; for example, if the primary antibody was mouse anti-human, then use goat anti-mouse IgG) for 1 h.
- Perform three quick rinses with 10 ml of PBS-Tween 20 (0.5% v/v) and then wash the membrane for 5 x 10 min with 10 ml of PBS-Tween 20 (0.5% v/v).
- After the last wash, transfer the membrane to a clean glass plate and cover the membrane with 10 ml of developing solution (for example ECL from Amersham International or Borhinger Manneihm) for 2 min.
- Drain the excess developing solution and wrap the membrane in SaranWrap. Fix it in an x-ray film cassette with the proteins facing up.
- Go to the dark room and expose an x-ray film for few seconds or up to several minutes.
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