Nondenaturing detergent eluants

Simple aqueous buffers of appropriate pH and sufficient Ionic strength are usually capable of preventing protein adsorption by Ionic interactions with the packing. However, certain proteins may still adsorb to packings by hydrophobic Interactions. This problem Is frequently observed with hydro-phobic membrane proteins, which also tend to be relatively insoluble in aqueous buffers thereby resulting In protein aggregation and anomolous behavior in SEC. 

Hydrophobic protein-protein or protein-packing interactions can be eliminated In many cases by the addition of detergents to the aqueous buffer. Protein solubilization without denaturatlon can be achieved with mild detergents such as sodium deoxycholate, Triton X-100, or Nonldet P40 (27). 

An Intrinsic limitation In SEC of proteins Is their natural variation In conformation and effective hydrodynamic volume. This variation affects the chromatographic properties of proteins and Is frequently responsible for deviations in the theoretical linear relationship between molecular weight and retention coefficients which can be obtained with synthetic polymers. 

This deviation may affect the separation potential In preparative SEC and It Is of primary concern when SEC Is used to determine protein siolecular weights. Strong protein denaturants, eg., Ionic detergents such as sodium dodecyl sulfate and chaotroplc salts such as guanidine hydrochloride, are known to convert native proteins to random coll conformations (28). Fish et al. (29) first reported that the use of denaturants to Induce uniform protein conformation greatly enhanced the resolution potential and accuracy of protein molecular weight calculations In conventional SEC. 

Apparently the protein resolution obtained In SDS Is markedly dependent 

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