The choice of the most appropriate rehydration solution for the sample will depend on its specific protein solubility requirements. A typical solution generally contains urea, nonionic or zwitterionic detergent, DeStreak™ Reagent or DTT, the appropriate Pharmalyte® or IPG Buffer (all available from Cytiva), and a tracking dye. The sample may also be included. The role of each component is described below, as well as the recommended concentration range.
Urea solubilizes and denatures proteins, unfolding them to expose internal ionizable amino acids. Commonly, 8 M urea is used, but the concentration can be increased to 9 or 9.8 M if necessary for complete sample solubilization.
Thiourea, in addition to urea, can be used to further improve protein solubilization, particularly for hydrophobic proteins (10, 16, 55–57). When using both, the recommended concentration of urea is 7 M and that of thiourea 2 M.
Detergent solubilizes hydrophobic proteins and minimizes protein aggregation. The detergent must have zero net charge—use only nonionic or zwitterionic detergents. CHAPS, Triton X-100, or NP-40 in the range of 0.5 to 4% are most commonly used.
DeStreak™ Reagent overcomes the problems of streaking that commonly occur due to reoxidation when running gels that contain basic regions above pH 7. The reagent stabilizes thiol groups such as disulfides, thus reducing streaking and extra spots caused by various oxidation stages of proteins (62). See section 2.6.2 (Using DeStreak™ Rehydration Solution) for more information and a protocol for use of this reagent.
DeStreak™ Rehydration Solution contains DeStreak™ Reagent, as described above. The Rehydration Solution also contains optimized concentrations of urea, thiourea, and CHAPS, and is ready for use after addition of the appropriate IPG Buffer.
IPG Buffer or Pharmalyte® (carrier ampholyte mixtures) improves separations, particularly with high sample loads. Carrier ampholyte mixtures enhance protein solubility and produce more uniform conductivity across the pH gradient without disturbing IEF or affecting the shape of the gradient. IPG Buffers are carrier ampholyte mixtures specially formulated not to interfere with silver staining following 2-D electrophoresis. Select an IPG Buffer with the same pH interval as the Immobiline DryStrip to be rehydrated (Table 17, Using DeStreak™ Rehydration Solution).
The advantages of increased concentration of IPG Buffer/Pharmalyte® are:
Higher concentrations of IPG Buffer/Pharmalyte® will limit the voltage usable during IEF and increase the time required for the focusing step.
Silver staining may require a prolonged fixing step to wash out carrier ampholyte that may cause staining background near the ion front of the second-dimension gel.
IPG Buffer or Pharmalyte® can be included in the stock rehydration solution or added just prior to use. The carrier ampholytes are included in the stock solution when multiple Immobiline DryStrip gels of the same pH range are to be used. Carrier ampholytes are added to single aliquots of the stock solution when the same stock solution will be used with different pH range Immobiline DryStrip gels.
Tracking dye (bromophenol blue) allows IEF progress to be monitored during the protocol. If the tracking dye does not migrate toward the anode, no current is flowing. Note: the dye migrates to the end of the strip well before the sample is focused!
Sample can be applied by including it in the rehydration solution. Up to 1 mg of sample per strip (dependent on the length of the strip and the pH range) can be diluted or dissolved in rehydration solution prior to IEF. The amount of sample required is dictated in part by the detection or visualization method used. For example, radiolabeling requires a very small amount of sample whereas Coomassie blue staining requires larger sample amounts.