World-2DPAGE Home
[Top] [Prev] [Next] [Bottom]

Immunoblotting

Immunodetection is a powerful and sensitive technique, which relies on the specificity of antibodies to identify single protein spots from 2-D PAGE. The technique we currently use for immunodetection protein identification is enhanced chemiluminescence (ECL). With this method, PVDF membranes are first stained to visualize proteins, following which the immunodetection is undertaken. This allows matching of proteins detected with ECL against those detected with the non-specific protein stain through computer comparison of both images. The mechanical strength of PVDF is also exploited as the same 2-D gel can be used many times for different antibodies. Immunoblotting is good to use where only small quantities of sample are available as it can detect as little as picogram amounts of protein, depending on the specificity of the antibodies. However, it is a slow technique, as it is only possible to identify a few proteins per gel per day. It also requires the prior existence of monoclonal or polyclonal antibodies, which may be expensive to make or obtain commercially. In the below protocol, we often probe with 8 or 9 antibodies at once to increase protein identification rates. However, in such cases we first check that there is sufficient pI and Mw difference in the proteins of interest to avoid identification ambiguities.

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.

  1. Block the membrane in 10 ml of a solution containing PBS (pH 7.2) and nonfat dry milk (5% w/v) for 30 min.
  2. 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.
  3. 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).
  4. 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.
  5. 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).
  6. 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.
  7. Drain the excess developing solution and wrap the membrane in SaranWrap. Fix it in an x-ray film cassette with the proteins facing up.
  8. Go to the dark room and expose an x-ray film for few seconds or up to several minutes.



[Top] [Prev] [Next] [Bottom]