Immobilized pH gradient strips

Olsson, I., Larsson, K., Palmgren, R. and Bjellqvist, B. (2002) Organic disulfides as a means to generate streak-free two-dimensional maps with narrow range basic immobilized pH gradient strips as first dimension. Proteomics 2, 1630-1632. 

Fig. 6. Two-dimensional polyacrylamide gel separation of hepatic proteins obtained from control and Entreated largemouth bass (injected IP with 2.5 mg/kg). The separation in the first dimension was on a pH 4-7 immobilized pH gradient strip and in the second dimension on an 8-16% gradient gel. A small segment of the gel is expanded for visual inspection. Arrows point to proteins that are differentially expressed in either E2 treated or control fish.

Fig. 1. 2D-PAGE. 180. ig of platelet proteins Is separated. For isoelectric focusing, immobilized pH gradient strips (pH 3-10) have been used.

Because the pi of a protein is based on its amino acid sequence, this technique has good resolving power. The resolution can be adjusted further by changing the range of the pH gradient. The use of immobilized pH gradient (IPG) strips has enabled reproducible micropreparative fractionation of protein samples, which is not consistently possible when ampholytes are used in the first dimension (Gorg et al., 2000). 

The introduction of the immobilized pH gradient (IPG strip) greatly enhances the potential of 2DE (Righetti and Bossi, 1997 Fichmann, 1999 Gorg et al, 2000). This is... 

For the first-dimension separation, a protein sample containing an appropriate cocktail of carrier ampholyte is applied to a standard immobilized pH gradient gel (IPG) strip and left overnight to rehydrate the strip. These strips are available in several narrow pH ranges for improved separation and are specially well suited for FFE-separated subfractions. Application of the appropriate electrical potential to the rehydrated strip results in separation of the proteins according to their intrinsic charge. 

Fig. 17.6. Isoelectric focusing diagrammatic view of isoelectric focusing (IEF) of a re-hydrated linear immobilized pH 3-10 gradient strip (Biorad, UK).

Gels create an "anchoring" point for buffering ions allowing immobilized (stable) pH gradients. And perhaps most important of all, stabilizers offer a convenient format for electrophoresis, where "strips" and "slabs" can be easily stained, destained, and otherwise manipulated in ways unthinkable for free solutions. Due to these significant advantages, virtually all electrophoresis is done with the use of a stabilizer. 

Fig. 1. Two-dimensional gel electrophoresis of culture supernatant proteins. Proteins were isolated from wild-type Streptococcus pyogenes strain NZ131 after 18 h of culture in 40 mL of chemically defined medium. Hundred micrograms of protein was separated with a 7-cm immobilized gradient strip (pH 4-7) (IPG GE Biosciences) and SDS-PAGE. Proteins were stained with SYPRO Ruby.

---