Proteomics/Protein Separations- Electrophoresis/IsoElectric Focusing
Isoelectric Focusing(IEF)
[edit | edit source]In Isoelectric focusing, proteins are separated by electrophoresis in a pH gradient based on their isoelectric point(pl). A pH gradient is generated in the gel and an electric potential is applied across the gel. At all pHs other than their isoelectric point, proteins will be charged. If they are positively charged, they will move towards the more negative end of the gel and if they are negatively charged they will move towards the more positive end of the gel. At its isoelectric point, since the protein molecule carry no net charge it accumulates or focuses into a sharp band.
Immobilized pH Gradeint (IPG) and IEF run
[edit | edit source]Immobilized pH gradients are used for IEF because the fixed pH gradients remain stable over extended run times at very high voltages. The pH gradients of IPGs are generated by means of buffering compounds that are covalently bound into polyacrylamide gels. IPGs are cast strips with plastic backing sheets and are commercially available in different pH ranges and lengths. They offer high resolution, great reproducibility, and allow high protein loads. Isoelectric focusing is run in the same solutions that are used to extract or solubalize the proteins. The IPG strips with the protein sample must be rehydrated in the rehydration/sample buffer during which protein samples are loaded into the strips. Rehydration can be active or passive. To load larger proteins active rehydration in small voltage is applied.As the sample is electrophorised, the proteins will migrate toward either the anode or cathode, depending on charge. The proteins will stop when they reach their respective pI. After the run in IEF cell, the proteins focus as bands on the strip according to their isoelectric points. The focused strips can be frozen for storage.