Isoelectric Point Fractionation
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Fractionation by Isoelectric Focusing
This technique separates intact proteins and peptides based on their isoelectric point (pI), which is the first step in a 2D gel electrophoresis project. The two approaches we utilize isolate the proteins/peptides in solution (Agilent OFFGEL) or in a protein chip (Protein Forest). Fractionated samples can then be further analyzed by 1D SDS PAGE or LC-MS/MS analysis (after digestion for proteins).
Isoelectric Focusing (Protein Forest)
Both intact proteins and peptides can be fractionated using the Protein Forest dPCTM Fractionator, Model SB-106. This instrument allows isoelectric focusing separation of peptides/proteins to 0.05 pH units using the dPC (digital ProteomeChip™). After separation, the gel plugs are digested or the peptides are eluted prior to LC-MS/MS protein identification. A Western blot can also be run for protein identification. The procedure concentrates the complex protein/peptide mixture > 3fold thereby, enhancing the amounts of the low abundance proteins/peptides which in turn, enhances the protein identification success rate.
OFFGEL Fractionation
Proteins and peptides are fractionated in the Agilent 3100 OFFGEL Fractionator which uses a novel isoelectric focusing technique. The fractions are isolated in solution, and thereby, the SDS PAGE separation can be avoided. Samples do require de-salting prior to further analysis.
- 0.1 – 0.6 pH resolution depending on consumable kit used
- In-solution fraction recovery
- µg to mg load capacity
