1. The IPG gel strip is rehydrated and seals tightly against the well frame
2. The diluted sample is equally distributed into all wells in the frame, and a cover seal is placed on top of the frame to prevent sample evaporation
3. A high voltage is applied to the ends of the gel strip and the protein or peptide molecules migrate through the gel until they reach a position where the pH equals the pI of the molecule
4. After fractionation, the separated proteins or peptides remain in the buffer solution and can be easily removed using a pipette for subsequent downstream processing
A closer look of the OFFGEL 3100 and its accessories:
In summary, the benefits of the Agilent 3100 OFFGEL Fractionator are:
- pI-based OFFGEL fractionation with liquid-phase recovery for easy transfer to downstream techniques such as LC/MS
- Reproducible fractionation with resolution as low as 0.1 pH for maximum MS sensitivity
- Compatibility with up- or downstream techniques such as immunodepletion, LC/MS or gel-based analysis for maximum flexibility
- pI values obtained act as additional validation parameters of MS hits and can be used to search for peptides with charged posttranslational modifications (PTMs)
- All additives can easily be removed after fractionation to avoid interference with nano-electrospray and MS detection
- Fractionation of up to 16 samples in parallel on two trays (eight samples each) for highest throughput
- Broad loading capacity from 50 μg to 5 mg of sample for analytical applications or maximum enrichment of low abundance proteins
- OFFGEL mode as well as conventional isoelectric focusing (IEF) based on immobilized pH gradient (IPG) gels
