DIGE substantially reduces protein spot variability caused by 2D-PAGE and increases detection of differentially expressed proteins.

A common method for quantifying proteins is to separate them by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and quantify the intensity of the spots with image analysis software. Protein spot variability can be quite high averaging about 60 to 80% for three biological replicates. Gel to gel variability is quite high requiring the researcher to make many technical replicates and select from the best of the gels made. Our lab decided to determine where most of this variability came from. We designed an experiment to determine if the variability came from the biological replicates, protein extractions or the custom made 2D-PAGE gels. A total of 27 gels were run; there were 3 gels of 3 extractions of 3 biological replicates of ‘Cabernet Sauvignon’ shoot tips. Nearly 500 common protein spots were quantified on all 27 gels. Variability was quantified with the coefficient of variation (CV). The average CV was highest for the gels and lowest for the biological replicates, averaging 55, 33 and 25% for gels, extractions and biological replicates, respectively. The CV ranged from 5 to 122% for some protein spots indicating that for many proteins it is difficult to get good quantitative data for comparative purposes. The variability of this method limits the number of proteins that can be quantified adequately and inflates the cost and labor due to the replacement of bad gels. A similar analysis was performed on buds of Seyval and Vitis riparia. The average CV of 861 spots was 68%. Difference Gel Electrophoresis (DIGE) is designed to reduce this variability. We found that DIGE significantly reduced the CV to about 33% and the use of internal standards reduced gel-to-gel variability and increased the detection of differentially expressed proteins