SDS protein complex

Both Reynolds and Karim worked at neutral pH, with denatured proteins, and with reduced disulfide bonds. Under these conditions, proteins are in a random coil conformation (Mattice et al., 1976), so that their hydrodynamic radius is monotoni-cally related to their molar mass. Takagi et al. (1975) reported that the binding isotherm of SDS to proteins strongly depends upon the method of denaturing disulfide bonds. Presumably, protein-SDS complexes are not fully unfolded when disulfide bonds are left intact, which breaks the relationship between molar mass and hydrodynamic... 

Jin LJ, Giordano BC, Landers JP. Dynamic labeling during capillary or microchip electrophoresis for laser-induced fluorescence detection of protein-SDS complexes without pre- or postcolumn labeling. Anal Chem 2001 73 4994-4999. 

The preparative SDS gel electrophoresis (see Section 1.3.1) is suitable for the purification of proteins, where the experimenter does not care about biological activity. The preparative SDS gel electrophoresis separates according to size. The protein s MW, which is known from (for example) cross-linking experiments (see Section 2.4), is used for the detection of the sought-after protein. After electrophoresis, the sought-after protein is available in the form of a denatured protein/SDS complex. Hence, the method makes sense as the last purification step (i.e., after purification steps that depend on charge differences or on activity have already been performed). 

Lyse the pellet with 1 or 2 ml of 1% SDS in TE. Complete the volume to 3.3 ml and add 3.96 g of solid cesium chloride. The final vol (4.25 ml) is suitable for the tubes of the Beckman SW50 and Kontron SP55.5 rotors (use tubes with small volumes avoid vertical rotors). A pre-run (10 min 10,000 rpm) allows to eliminate proteins-SDS complex floating on top. Then, adjust the refractive index to 1.399-1.400 (density 1.7 g/cm ). 

PAGE in the presence of sodium dodecyl sulfate (SDS) Most proteins bind the anionic detergent SDS in a constant mass ratio, 1.4 g of SDS per gram of protein, effectively masking the intrinsic charge of the protein. Consequently, the charge-to-mass ratio of almost all protein-SDS complexes is constant, their mobilities in a free solution are identical, and they can be electrophoretically separated only in... 

Modern cross-linked agarose matrixes (82) may be yet less charged than other polysaccharide-based packings. A recent report (83) implies that KsEC protein-SDS complexes is insensitive to buffer concentration over the range 0.03 M - 0.1 M on such agarose-based gels. 

In a free solution in the presence of SDS, proteins acquire almost the same zeta potential (which is a function of the net surface charge density) and accordingly migrate at the same speed [39] in the capillary channel. When capillary electrophoresis of protein-SDS complexes is performed in polyacrylamide gels (sieving gels), the complexes migrate with a velocity dependent only on the size of the complex [92, 93]. 

Ion-retardation resins, which consist of acrylic acid polymerized inside a strong anion-exchange resin on a polystyrene divinylbenzene matrix [30], are also effective for removal of SDS from proteins. Passage of a protein-SDS complex through the resin results in complete retention of SDS and elution of protein with 80-90% recovery [31]. The capacity of the resin for SDS is more than 2.2mg/g, which effectively reduces the SDS level to less than one molecule of SDS per protein molecule. Because SDS binds tenaciously to the resin, it cannot be removed and the resin must be discarded after use. In the presence of buffers, adsorption of SDS by an ion-retardation column is reduced, resulting in incomplete removal of detergent from the protein. This can be circumvented by prior removal of buffer by SEC or, more conveniently, by the addition of a few grams of size exclusion gel to the head of the ion-retardation resin bed to retard the buffer [4]. 

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