The IPG strips need to be rehydrated with the labeled sample, which is mixed with the respective IPG buffer. The sample is then placed over the IPG strip and rehydration is allowed to occur by incubating the strips at RT for 16 hrs. After 1 hr, the samples are overlaid with mineral oil to prevent sample evaporation. The rehydrated strips are further used for isoelectric focusing (IEF). In IEF, the rehydrated strips are first placed in the IEF tray and program for IEF run is selected as per the strip length and pH range. In IEF, the applied voltage gradient brings about separation of proteins on the basis of their isoelectric point (pI), at which the net charge on a given protein is zero.

C. Second Dimension (SDS-PAGE)

The separation of proteins on the basis of their molecular weight is brought about in the Second dimension by SDS-PAGE. The focused strip is run on the gel in a direction, which is orthogonal to the IEF run. Follow previous lecture for more details of this step.

D. Scanning and Image Acquisition

After the second dimension run is completed, the gels are scanned at 3 different wavelengths, which are the excitation wavelengths of each of the 3 dyes, namely Cy3, Cy5 and Cy2.  The resulting images are stored for further analysis. `

E. Image Analysis and Data Interpretation

The saved images are further analyzed by using software especially designed for 2D- DIGE, such as DeCyder. The pair-wise analysis between test and control samples can be performed using Differential In-gel Analysis (DIA) module, whereas the analysis between multiple samples belonging to two different groups can be performed using the Biological Variation Analysis (BVA) module of the software. Details of the data analysis will be described in next lecture.