Protein Protocols Protein Protocols

170 Gianazza
Fig. 1. Structure of the IPG matrix. Different regions of an IPG made up with a basic and an
acidic Immobiline are schematically drawn from cathode to anode. In each region, pH depends
from the concentration ratio between the basic and the acidic dissociating groups. From the
Henderson-Hasselbalch equation, where the concentration of the basic monomer is twice the
concentration of the acidic monomer, pH equals the pK of the base, where the concentration of
the basic monomer is one-half the concentration of the acidic monomer pH equals pK of the
acid, and where the two concentrations are alike, pH equals the mean between the two pKs.
main one is the variability of the analytical needs. 2-D protocols usually aim at the
resolution of all peptide components of a complex mixture. Although the proteins in
cellular extracts and biological fluids have mostly mildly acidic pI values, this is not
true of all samples, and the optimal pH course should be devised accordingly. Moreover,
after specific qualitative or quantitative variations have been detected for some
spots as the result of a given experimental treatment, it is usually worth investigating the
area of interest under conditions of maximal resolution. This is especially true if
the spots have to be identified or characterized, e.g., by mass spectrometry (MS) techniques
after a blotting step (7). IPGs allow the tailoring of wide, narrow, or ultranarrow