LOCALIZATION OF IMMUNOREACTIVE
2D GEL SPOTS FOR EXCISON
AND PROTEIN IDENTIFICATION

1. For 2D Western blot analysis, partially transfer proteins in 2D gels to PVDF or nitrocellulose membranes (eg, at 320 mA/gel for 25 min using a BioRad Semi Dry Transfer Cell). Visualize proteins remaining in the gel with colloidal Coomassie blue or silver.
   
   
2. To facilitate localization of immunoreactive spots in the stained gel, transfer the 2D Western profiles from the membrane to a transparency with an office copy machine (eg, Xerox copier).   
   
   
3. Enlarged the transparency to the size of the swollen gel after staining/destaining. To determine how much to enlarge the transparency, mark/measure gel dimensions on the PVDF membrane during blotting and compared with gel dimensions after staining/destaining.
   
   
4. Overlaying the Western transparency on the stained gel to correlate immunoreactivity with specific gel spots.