How are the 2D densitometry images made?
The Symansis product was focused on a pH 3-10 gradient IPG strip and then run on a linear gradient SDS-PAGE gel along with and a set of molecular weight markers. The gel was stained using Deep Purple or Coomassie Blue protein stains. The relative intensities of the proteins spots visible on the 2D-gel were analysed using the software ImageJ software(http://rsb.info.nih.gov/ij/). Densitometric analysis was performed on the spots within a selected area of the gel and a background subtraction was conducted using the appropriate blank region of the gel (region lacking protein spots). Volume integration was performed on each protein spot of interest, and from this the centre of mass and its relative percentage intensity were calculated. The molecular weights and pI values of the respective spots were determined by comparing them to the position of the precision MW markers and the linear IGP strip. The former were fitted to an exponential function with a 4th order polynominal to interpolate
