IEF/SDS-PAGE

Neukirchen et al. (1982) from the Max-Planck Institute employed a similar miniaturized IEF/SDS-PAGE system that was roughly 2 cm x 2 cm. Silver staining was used to detect spots containing as little as 10 pg of protein, and electrophoresis was used to separate the proteins contained within a single Drosophila egg. 

Peptide mapping, HPLC, IEF, mass spectrophotometry, amino acids sequencing Peptide mapping, HPLC, mass spectrophotometry IEF, SDS-PAGE, HPLC, amino acids sequencing SDS-PAGE, HPSEC... 

Holen, E., Elsayed, S. 1990. Characterization of four major allergens of hen-egg white by IEF/-SDS-PAGE combined with electrophoretic transfer and IgE-immunoautoradiography. Int Arch Allergy Immunol 91 136-141. 

Moreover, experimental reference maps of human tissues were studied p7 and Mr coordinates of identified spots were retrieved from the SWISS-2D-PAGE database, the values <3X = 0.009 pH and av = 0.0002 log Mr were assumed for spot dimension since they represent the standard case for experimental 2D-PAGE maps—normal sample loading of a tissue homogenate (ca. 1 mg total protein) and standard gel sizes (18 x 20 cm, IEF x SDS-PAGE). 

Poehling reported a microscale two-dimensional gel electrophoresis system 25 years ago (Poehling and Neuhogg, 1980 Neuhoff, 2000). In that system, separation in the IEF dimension was performed in thin, gel-filled tubes. After IEF, the gel was transferred to a 3 cm x 3.5 cm polyacrylamide gel, where proteins were separated by SDS-PAGE. Several hundred components were resolved from a few micrograms of protein homogenate. 

Whitesides reported a microfabricated device where isoelectric focusing was performed in a horizontal channel and SDS-PAGE was performed in a series of vertical channels machined at right angles to the IEF channel (Chen et al. 2002). 

In 1956, Smithies and Poulik first used 2-DE combining paper and starch gel electrophoresis to separate serum proteins. Nearly 20 years later, polyacrylamide was applied as a support medium. Charge-based protein separation followed as isoelectric focusing (IEF), applied to SDS-PAGE. Later, urea and nonionic detergents were used in IEF-2DE. The most significant achievement was the separation of proteins from E. coli. 

The separation length is the most significant factor influencing the resolving capacity of he gel. Application of IPG IEF gel of 18 cm and second-dimension SDS-PAGE of 20 cm long allows resolution of a complex mixture with approximately 200 proteins. Rapid screening can be achieved by mini-gel formats. However, only a few hundred proteins can be separated by such system. So, the amount of the protein separated depends upon the size of the gel. 

Since, the broad protein and carbohydrate band overlapped each other in the 42 - 200 kDa range of SDS-PAGE and in the pH 3.0 - 4.3 range of IEF-PAGE (data not shown), MALDI MS of ARS2 was performed as previously reported for hapten-carrier protein conjugates For more detail MW determination [29-31],... 

For isoelectric focusing (IEF) and SDS-PAGE gels, materials are those descnbed by O Farrell (1,2) and Laemmli (3). It should be noted that for IEF, acrylamide and Zus-acrylamide must be of the highest level of purity, and urea must be ultrapure (enzyme grade) (see Methods in Molecular Biology, Volume 3, Chapters 15-21). 

Polyacrylamide gel (SDS-PAGE, IEF, native PAGE, or Tris-tricine SDS-PAGE) containing separated proteins (see Basic Protocols 1 and 2 see Alternate Protocols 1 to 4)... 

Whereas SDS-PAGE and other discontinuous techniques are generally quite tolerant of sample impurities and buffer and ionic variations, the quality of the sample and the nature of the solution it is loaded in have a strong influence on the quality of an IEF separation. The sample must be as free as possible of salts, buffers, and other small charged molecules,... 

IEF has a similar resolving power to SDS-PAGE, but it has less applicability due to the limited solubility of many proteins under IEF conditions. 

The third example concerns an industrially relevant enzyme, glucose isomerase (see Chapters 7,10, and 19). Table 8.6 reproduces the purification data, and Figure 8.10 shows the SDS PAGE and IEF (isoelectric focussing) diagrams. 

Figure 8.10 SDS-PAGE for the determination of subunit molecular mass and IEF for the isoelectric point of xylose isomerase from Thermoanaerobium. Source Liu (1996).

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