2-DE provides a method for separating complex mixtures based on two intrinsic properties of proteins: their isoelectric points and molecular weights. In the first dimension, termed isoelectric focusing (IEF), the proteins are separated out on a thin strip of polyacrylamide gel according to a specified pH gradient. The length of the strip and the range of the immobilised pH gradient (IPG) can be chosen to suit different types of samples. In the second dimension the proteins are separated according to their molecular weight. The resulting 2-DE can be stained or transferred for blotting applications. Specialised visualisation techniques can be used to detect changes in protein expression between two samples, referred to as Difference In-Gel Electrophoresis (DIGE) or to investigate certain post-translational modifications.
Efforts should be made to minimise the amount of salts within protein samples. Samples that contain high salt levels require extra processing prior to 2-DE. Cell lysates should be treated with DNase and RNase. The amount of protein required will depend on the purpose of the investigation and should be discussed with the APC.
Certain sample types will require a clean-up step prior to 2-DE. A protein assay may also be performed to ensure the appropriate amount of protein is applied to the IPG strip. The 2-DE process can take up to three days. Staining may take an additional day, depending on the method used. Stains typically used are Coomassie R250, colloidal Coomassie or silver.
Protein spots may be excised for identification by tryptic digest and mass spectrometry.
The APC has two IEF platforms for separation in the first dimension: an IPGphor II and Multiphor II. Depending on the strip length, SDS-PAGE in the second dimension can be performed using the Ettan DALTtwelve system (capacity: 12 large-format gels), the SE 600 Ruby system (capacity: 6 medium-format gels) or the miniVE system (capacity: 6 mini-gels). If required, electro-transfer can be carried out for large, medium and mini-gels with the TE77 PWR semi-dry apparatus. All equipment is from GE Healthcare.
The resulting gels will be digitally scanned with the ImageScanner, DIGE Imager or Typhoon fluorescence scanner (all from GE Healthcare). If required, 2-DE gel image analysis can be performed using the software packages ImageMaster Platinum or DeCyder (both GE Healthcare).
A written report is provided, describing the analysis undertaken and outlining the evidence for a protein's identification. If no hit is obtained, an indication of the possible reasons may be included. The report is normally sent by email, as PDF. Raw data are in proprietary formats and are not included with the report. Data are archived by the APC.