Fractionation range, proteins

Type Bead size djo (/xm) Size exclusion pore dimension (nm) Exclusion limit protein (M,) Fractionation range protein (/V ,) pH stability (long term/ short term)... 

Sephadex type Grade Dry bead diameter (/urn) Fractionation range peptides and proteins (g/mol) Fractionation range dextrans (g/mol) Swelling factor (ml/g dry Sephadex) Maximum operating pressure" (cm H,0) Permeability Ko Maximum linear velocity" (cm/hr) Swelling time (h) ... 

Shifts in the SEC fractionation range are not new. It has been known for decades that adding chaotropes to mobile phases causes proteins to elute as if they were much larger molecules. Sodium dodecyl sulfate (SDS) (9) and guanidinium hydrochloride (Gd.HCl) (9-12) have been used for this purpose. It has not been clearly determined in every case if these shifts reflect effects of the chaotropes on the solutes or on the stationary phase. Proteins are denatured by chaotropes the loss of tertiary structure increases their hydrodynamic radius. However, a similar shift in elution times has been observed with SEC of peptides in 0.1% trifluoroacetic acid (TEA) (13-15) or 0.1 M formic acid (16), even if they were too small to have significant tertiary structure. Speculation as to the cause involved solvation effects that decreased the effective pore size of the... 

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). 

Subsequent work by Gardiner et al. showed that in a relatively complex mixture like human serum, the association between a metal and protein or ligand could be said to have been established when their Gaussian distribution coincide. This is more likely to be true if the elution volumes of the constituents are in the fractionation range rather than in the excluded volume. Examples of the usefulness of immunological techniques as an aid in identification of proteins are given. Necessary clean-up procedures are also suggested. 

Because of the very high molar masses of enzymically polymerized lignin sulfonates, Sephacryl S-300 is used as the gel matrix. The fractionation range is 10 000 to 1 000 000 dalton. Even in this case proteins of known molar mass can be used as calibration standards (Figs. 7 and 8). 

Name Fractionation Range for Proteins (daltons) Water Regain (mLVg dry gel) Bed Volume (mLVg dry gel)... 

Figure 7.11. The effects of exposure temperature on protein synthetic patterns of isolated gill tissue from specimens of 13°C-acclimated Tegula funebralis. Autoradiographic images illustrate newly synthesized (35S-labeled) proteins of several size classes (molecular mass standards are shown in the left lane). Two specimens from each temperature of incubation are shown. At temperatures above 24°C, synthesis of heat-shock proteins in the molecular mass ranges of 38, 70, 77, and 90 kDa is induced. Hsp synthesis becomes an increasingly large fraction of protein synthesis as exposure temperature increases, and by 38°C, only synthesis of hsp70 is observed. By 39° C, no protein synthesis takes place. (Figure modified after Tomanek and Somero, 1999.)...

As noted in Section VIII, preparative ion-exchange and hydrophobic interaction chromatographic procedures have come to occupy, from practical considerations, central positions in the fractionation of protein mixtures, typically enabling purification factors in the range of 10- to 100-fold.6,364,366... 

Gel filtration media are characterized by their exclusion limit and their fractionation range for macromolecules. Therefore the most important selection criterion to choose among gel filtration media is the range of molecular mass of the proteins that have to be separated. For IgG polishing, the solid phase of choice should have an exclusion limit of 300 KDa for IgM the exclusion limit should be of about 1200 KDa and for Fab fragments it should be of 80 KDa. 

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