Chaotropic extraction buffers that preserve the native charge of proteins making them suitable for sample extraction for 2D gels
One of the most important considerations before running 2D gel electrophoresis is the choice of protein solubilization buffers. A suitable buffer must solubilize proteins effectively, without disturbing the native charge of the proteins.
Urea, a common chaotrope, is widely used for solubilization and denaturation of proteins. One of the disadvantages of using urea is carbamylation. In water, urea exists in equilibrium with ammonium cyanate, the level of which increases with increasing temperature and pH. Cyanate reacts with α-amino and ε-amino groups of proteins and induces a change in the isoelectric point of proteins. This leads to artifactual results. Therefore, carbamylation must be avoided.
One way to minimize the risk of carbamylation is to prepare the urea-based reagents fresh before each use. G-Biosciences has developed a series of dry, urea-based, pre-mixed and ready-to-use solubilization buffers. Simply add an appropriate volume of the supplied rehydration buffer to the dry buffer. Then use to solubilize proteins, saving time and improving the quality of IEF/2D gel electrophoresis (Patent Pending).
FOCUS™ Extraction Buffers are also designed to be used as rehydration buffers for IPG strips.
FOCUS™ Extraction Buffers are experimentally optimized concentrations of critical, buffering and stabilizing agents, including urea, thiourea, Nonidet® P-40, CHAPS and sulfobetaines. The FOCUS™ Extraction Buffers are designed to produce optimal protein extraction and improved spot resolution for 2D gel analysis.
A range of FOCUS™ Extraction Buffers have been developed. Depending on the nature of the samples, one or more of the buffers suitable for your applications can be ordered. FOCUS™ Extraction Buffer-I is suitable for most applications; however, for stronger solubilization effects, we recommend FOCUS™ Extraction Buffer-II, -III, -IV, -V or -VI. A trial kit that contains all six FOCUS™ Extraction Buffers and each of their corresponding diluents is also available. The trial kit is designed for those who prefer to experiment for establishing a suitable extraction and solubilization buffer for their protein samples.
The following FOCUS™ Extraction Buffers, listed with their major components, are available:
| Major Components |
| FOCUS™ Extraction Buffer I | Urea & Nonidet® P-40 |
| FOCUS™ Extraction Buffer II | Urea & CHAPS |
| FOCUS™ Extraction Buffer III | Urea, thiourea, CHAPS & amidosulfobetaine (ASB)-16 |
| FOCUS™ Extraction Buffer IV | Urea, thiourea, CHAPS & sulfobetaine (SB) 3-10 |
| FOCUS™ Extraction Buffer V | Urea, thiourea & CHAPS |
| FOCUS™ Extraction Buffer VI | Urea, thiourea, CHAPS & non detergent sulfobetaine 201 |
For analysis of a proteome, a single buffer may not be suitable and sequential solubilization using different FOCUS™ Extraction Buffers will help in identifying more and new proteins (see figure 1). See the application note in the technical section below for more information.
Features
- Convenient & easy to use: simply hydrate and use
- Prevents urea-induced protein carbamylation
- Prevents waste of unused reagents
- No artifactual protein bands due to dust & human skin contamination
Applications
- Optimal protein extraction & solubilization for 2D gel electrophoresis & other applications
- Rehydration buffer for IPG-strips
- Improved spot resolution for 2D gel analysis