Sample preparation is a critical step in 2D gel electrophoresis and is key to the success of your experiment. Many factors play an important role in designing a procedure for sample preparation. We offer products to help you be successful including:
The 2D Electrophoresis Sample Rehydration Buffer, also as known as the sample buffer, is used to denature and solubilize protein samples, and rehydrate the IPG strips. The first step in sample preparation is selecting and/or preparing a suitable sample rehydration buffer. Due to the large variety of proteins, there is no universal sample rehydration buffer. The starting material and goal of the experiment are two factors to consider when selecting a buffer. The sample rehydration buffer must:
- Maintain proteins in solution during rehydration of the IPG strips and IEF
- Not have any effect on the pI of the protein
The buffer typically contains a denaturing agent (urea or urea/thiourea), solubilizing agent (non-ionic or zwitterionic detergent and ampholytes), and reducing agent (DTT). The table below lists the major components of the sample rehydration buffer, their function, and their recommended concentration. Note that the final concentration must be optimized based on the solubility of your proteins. Optimization is usually achieved by varying the concentrations of detergent, urea, ampholytes, and reducing agent.
2D Electrophoresis Rehydration Buffer Components
|Urea||Denatures and solubilizes proteins||• 8 M urea or 9 M urea in some cases|
• For urea/thiourea solution, use 5–8 M urea with 2 M thiourea
|• Urea solutions are prepared fresh or stored frozen at –20oC. |
• De-ionize urea solutions on a mixed-bed ion exchange resin using manufacturer’s recommendations.
• Thiourea is used to increase solubility of some proteins (Rabilloud, 1998)
|Detergent||Solubilizes the proteins and helps to maintain the proteins in solution during rehydration and IEF||Total detergent concentration range is 0.5–4%||• Use non-ionic or zwitterionic detergents such as CHAPS, NP-40, CHAPSO, and sulfobetaines (SB3-10) Chevallet et al., 1998).|
• Ionic detergents such as SDS are not recommended.
|Reducing Agent||Cleaves the disulfide bonds in the proteins||DTT or DTE at 20 mM to 100 mM||• Use DTT or DTE (dithioerythritol). |
• β-mercaptoethanol is not recommended for reduction (Righetti et al., 1982).
|Ampholytes||Helps in solubilizing proteins and aids in maintaining the pH gradient 0.2–2%||Note: Higher ampholyte concentration requires longer focusing times.||• Use the appropriate carrier ampholytes based on the pH range of the IPG strip.|
• May use ampholytes pH 3–10 for all pH ranges of IPG strips.
Using Carrier Ampholytes Versus IPG Strips
IEF is performed in tube gels (polyacrylamide gels cast in long glass tubes) using carrier ampholytes. The protein sample is applied to the gel and IEF is performed at high voltage. Once the current is applied, the carrier ampholytes form a continuous pH gradient. While the gradient is formed, the protein migrates in the pH gradient to its pI.
At the end of IEF, gel is extruded from the tube and equilibrated in buffer containing SDS to prepare the proteins for migration into the second dimension SDS gel. The tube gel is placed on top of a polyacrylamide gel containing SDS and subjected SDS-PAGE.
Disadvantages of Carrier Ampholytes
Using immobilized pH gradient (IPG) gels for first dimension IEF eliminates the problems associated with carrier ampholytes. The immobilized pH gradient (IPG) gel is formed by casting polyacrylamide gels using acrylamido buffers (acrylamide derivatives containing charged groups) on a plastic backing. Since the pH gradient is cast into the gel, the gradient is more stable and reproducible. After the IPG gel is polymerized, the gel is washed, dried, and cut into narrow strips (IPG Strips).
The pH gradient on the IPG strip can be linear or nonlinear (NL). The non-linear gradient is usually expanded from pH 4-7 resulting in a sigmoidal pH gradient. The non-linear IPG strips are useful for analyzing samples containing many proteins with pI in the range of 4-7, which is typical for most crude lysates from all species.
Advantages of IPG Strips
2D Electrophoresis Running Buffers & Focusing Buffers
Another critical component of the 2D electrophoresis system is the running buffer as well as the focusing buffers. Invitrogen offers convenient and reliable solutions for a better performance.
ZOOM® IPGRunner™ System Technical Guide
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.