Polyacrylamide gels fractionation ranges

TABLE 16.8 Producers (Bio-Rad) Specification of Fractionation Ranges of Polyacrylamide-Based Bio-Gel P Gels... 

Prokaryotic cells express hundreds to thousands of proteins while higher eukaryotes express thousands to tens of thousands of proteins at any given time. If these proteins are to be individually identified and characterized, they must be efficiently fractionated. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has typically been use to study protein mixtures of <100 proteins. Onedimensional electrophoresis is useful because nearly all proteins are soluble in SDS, molecules ranging from approximately 10,000 to 300,000 molecular weight can be resolved, and extremely basic or acidic proteins can be visualized. The major disadvantage to one-dimensional gels is that they are not suitable for complex mixtures such as proteins from whole cell lysates. 

Figure 15.5 IEF gel electrophoresis of formalin-treated RNase A (a) and its fractions (b) separated by gel filtration, (a) M, IEF markers (pi) lane 1, unfractionated formalin-treated RNase A. (b) lane 1, monomer lane 2, dimer lane 3, trimer lane 4, tetramer lane 5, pentamer. IEF gel electrophoresis was performed using precast 5% polyacrylamide gels with a pi range of 3-10. The pi range for the individual oligomers in 15.5b appears greater than that for the unfractionated sample because a higher concentration of protein was used for the lanes in 15.5b. This results in an increased staining intensity for the oligomers at the pi extremes, which are minor components of the unfractionated sample in 15.5a. See Rait et al.10 for details.

The exclusion gels discussed in Section F are not appropriate for HPLC use because they are soft and, except for small-pore beads (G-25 and less), collapse under high-pressure conditions. Semirigid gels based on cross-linked styrene-divinylbenzenc, polyacrylamide, and vinyl-acetate copolymer are available with various fractionation ranges useful for the separation of molecules up to 10,000,000 daltons. 

Similar materials are produced by other manufacturers. For example Bio-Rad offers the Bio-Gel-P range of polyacrylamide material (P-2 to P-100) in various parti-de sizes (diameter) extra fine (< 45 pm), fine (45-90 pm) and medium (90-180 pm). The Bio-Gel-A range of agarose (A-0.5 m - A-150 m) is likewise available in a range of particle sizes fine (38-75 pm), medium (75-150 pm) and coarse (150— 300 pm). Table 4-1 lists a range of recommended gel filtration material, together with details of particle size and effective fractionation ranges. 

Figure 2 illustrates the fractionation of a complex sample, an extract from Bermuda grass, which is a cannon allergen. Because of the heterogeneity of the sample, a broad range Ampholine was used, pH 3.5 - 10. The results of the fractionation were assessed by analytical polyacrylamide gel isoelectric focusing. Shown are the patterns of the original mixture and of the ten RIEF fractions. As can be... 

Ing cellulose acetate strip. This Instrument Is marketed by LKB Pro-dukter under the name Tachofrac. Kobayashl et al. (30) have also described preparative capillary ITP experiments using a syringe to extract fractions after migration across a potential gradient detector. Hjert n (31) has described a micropreparative zone electrophoretic technique In which the separation takes place In a capillary filled with a polyacrylamide gel. A flow of buffer past the end of the capillary sweeps the samples through a UV detector after which collection Is possible. This last method would seem to be the least useful because the smaller diameter tube limits the protein capacity to the ng to ug range. 

---