IEF and 2-D Standards

IEF standards allow dependable and reproducible pI calibration in IEF gels. IEF standards consist of a mixture of nine native proteins with isoelectric points ranging from 4.45 to 9.6. Five of the nine proteins are naturally colored to provide continuous monitoring of the focusing process. IEF standards are for use in analytical acrylamide and agarose isoelectric focusing gels under native conditions. The mixture is provided in a stable aqueous solution, and no reconstitution or dilution is required prior to use.

Constituent Proteins of IEF Standards* Protein Color pI 1. Cytochrome c Red 9.6 2. Lentil lectin (3 bands) — 7.8, 8.0, 8.2 3. Human hemoglobin C Red 7.5 4. Human hemoglobin A Red 7.1 5. Equine myoglobin (2 bands) Brown 6.8, 7.0 6. Human carbonic anhydrase — 6.5 7. Bovine carbonic anhydrase — 6.0 8. β-lactoglobulin B — 5.1 9. Phycocyanin (3 bands) Blue 4.45, 4.65, 4.75 * Because the IEF standards are in native form, they cannot be used with reducing or denaturing agents such as urea, β-mercaptoethanol, or dithiothreitol. For calibration or IEF tube gels containing urea, use 2-D SDS-PAGE standards.

IEF standards strained with IEF gel stain. Run on Criterion™ (left) and Ready Gel™ IEF gels (right).

IEF standards for accurate pI calibration of native IEF gels. A. 5 µl of the standards were stained with Coomassie Blue R-250 dye and crocein scarlet B. 5 µl of the standards were left unstained

These unique protein standards calibrate the pI and molecular weight of the proteins in 2-D SDS-PAGE applications. The standards consist of seven reduced, denatured proteins that can be run internally on the same gel as the sample, or alone to test the ampholyte mixture and the reproducibility of the gels. Protein spots can be visualized with silver or Coomassie Blue staining. No dilution of the standards is required.

Two-dimensional electrophoretic protein pattern of 2-D SDS-PAGE standards. The standards (2.5 μl) were run on 7 cm ReadyStrip™ IPG strips, then in the Mini-PROTEAN^® II cell. For method details, see Klose (1975), Klose and Feller (1981), and Jungblut and Seifert (1990). The gel was stained with Bio-Rad’s silver stain kit.