Sdspage and IEF Gels

For SDS-PAGE gels (or IEF gels), a MW or pI standard is normally run in one lane. The following procedure is used to identify the immunoreactive band and determine its MW or pI.

1. Scan the ECL-developed film, the Coomassie-stained nitrocellulose membrane, or a lane of the Coomassie-stained gel (in which standard proteins or the sample were separated) with a computing densi-tometer of sufficient resolution.

2. Use a graphics program to make the images the same length (all were originally from the same gel and are therefore of the same size, but nitrocellulose shrinks when stained).

3. When the images are perfectly aligned, the immunoreactive band can be distinguished in the Coomassie stained lane and its MW or pI determined by comparison with the appropriate lane.

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