Protein samples loaded on iso-electric focusing (IEF) gels should ideally have low conductivity and be free from agents known to interfere with net protein charge. Protein samples containing interfering agents, including ionic detergents, metal ions, salts, lipids, charged polysaccharides, peptides, nucleic acids, enzyme substrates, substrate analogs, inhibitors, plant products (phenols, etc.) and other agents having a charge, routinely result in smeared or low resolution gels. On the other hand, when a protein solution is dilute, it may be difficult to load an appropriate amount of protein onto the gel without concentrating the protein solution first.
Perfect-FOCUS™ is based on quantitative precipitation and concentration of protein solutions using UPPA™ (Universal Protein Precipitation Agent) and has been specifically developed for preparing lower conductivity protein samples for iso-electric focusing gels. Perfect-FOCUS™ is designed to clean and remove interfering agents from protein samples and concentrate protein solutions that give poor protein spot resolution during 2D electrophoresis.
Protein solutions as dilute as 1ng/ml, if treated with Perfect-FOCUS™, can be quantitatively precipitated into a small volume. Protein precipitation is not affected by the presence of detergents or other common laboratory agents. After precipitation, the precipitate is washed to remove salts and other non-protein interfering agents. At this stage, the protein pellet is ready to be solubilized and reconstituted in a small volume of the sample loading buffer and then loaded onto electrophoresis gel for perfect protein migration patterns.
The collected protein sample has a conductivity of approximately 40 to 50µS and is substantially free from non-protein agents, making the protein sample ideal for critical IEF and 2D gel electrophoresis studies, with improved spot resolution and greatly reduced spot streaking. If the protocol is followed correctly, the recovery is generally 100%. Perfect-FOCUS™ treated protein samples are compatible with mass spectrometry analysis and show identical peptide spectra.
Features
After treatment with Perfect-FOCUS™:
- Conductivity lower than 50µS
- Samples free from non-protein interfering agents
- Greatly reduced spot streaking and improved spot resolution
- Perfect protein migration patterns
- Suitable for running 2D gels and compatible with mass spectrometry analysis
- Process up to 50 protein samples (or 6 with the trial size kit), 1-100µl/each
Applications
- Prepare low conductivity samples for IEF (isoelectric focusing) and 2D gel electrophoresis applications
- Concentrate and clean protein samples
- Remove interfering agents from protein solutions
- Improve protein spot resolution and prepare streak-free 2D gels
