Detergents octyl glucoside

Many protein samples require the use of detergents for their solubilization. For IEF work, the zwitterionic detergents CHAPS and CHAPSO, or the nonionic detergent octyl-glucoside, at concentrations of 1-2% in the gel are recommended. Even in the presence of detergents, some samples may have stringent salt requirements and may aggregate in salt-free environments. 

A similar activity level was obtained in the deoxycholate, Triton X-100, and NP-40 extract preparations. Octyl glucoside and CHAPS extract preparations showed no detectable prephenate aminotransferase activity. When the hemoglobin step was used, there was no increase in the soluble activity recovered in the initial supernatant fraction, but the specific activity of the deoxycholate (the only detergent tried in this experiment) extract increased about tenfold. We would anticipate equally good results with use of Triton X-100 or NP-40 in combination with the hemoglobin step. 

S.A. = 0.67 in octyl glucoside buffer) could be found in all of the detergent-extract preparations. As above, the hemoglobin step increased dramatically the S.A. of shikimate dehydrogenase assayed in detergent-extract preparations. 

Dissolve 5.04 g urea (final 8 M) and 788 (J.1 carrier ampholyte mixture (e.g., Pharmalyte Amersham Pharmacia Biotech final 7.5%) in water to 10.5 ml. Add a reducing agent (60 mM dithiothreitol) and/or a detergent (0.5% [w/v] Triton X-100, CHAPS, or octyl glucoside), if desired. Make fresh. 

The ideal sample run on the Rotofor cell would contain only the protein mixture, water, and ampholytes or buffers. However, pi precipitation may require that 3 M urea be included for solubility. When higher urea concentrations are needed, the Rotofor cell is run at 12°C. Detergents (1-2% w/v) may also be added to samples. Zwitterionic detergents, such as CHAPS, CHAPSO, and nonionic octyl-glucoside are satisfactory. 

None of the poly-amino acids were found to be very soluble in any of the above mixtures. DGK3M was observed to be partially, but incompletely soluble in the SDS, dodecyl trimethylammonium, and octyl glucoside mixtures. The general difficulty of solubilizing these polypeptides in traditional denaturant and detergent solutions may reflect the inability of the solutions to overcome a kinetic barrier to disaggregation and should not necessarily be taken to imply thermodynamic instability of the fiiUy dispersed proteins in the solutions. 

Many different methods [340] are available for the reconstitution of bacteriorhodopsin into liposomes (a) sonication of purple membrane with dried, dispersed lipids [341] (b) co-precipitation of lipids and bacteriorhodopsin from organic solvents, such as dimethylsulfoxide [340] (c) dispersion of purple membrane sheets or monomeric bacteriorhodopsin and lipids in detergents, such as Triton X-100 [340], cholate [17,325], deoxycholate [170], or octyl-glucoside [327,342], followed by dialysis or removal of the detergent with Bio-Beads. 

Figure 5. Comparison of native and refolded rabbit muscle CK incubated with octyl glucoside extracts. Lanes 1-4 (10% SDS-PAGE) 1.2 gl each of native rabbit muscle CK incubated for 3 days without (Lane 1) or with 133 pi (Lane 2) or 33.3 pi (Lane 4) octyl glucoside extract supernatant 133 pi octyl glucoside extract pellet (material which precipitated out of the octyl glucoside extract when it was washed with 50 mM Tris to remove detergent and concentrated 20-fold) (Lane 3). Lanes 5-8 2 pi native rabbit muscle CK refolded without (Lane 5) or with 133 pi (Lane 6) or 33.3 pi (Lane 8) octyl glucoside extract supernatant 133 pi octyl glucoside extract pellet refolded from the precipitated material described for Lane 3 (Lane 7).

During the solubilization of membranes, the purification of integral membrane proteins, and the reconstitution of membranes, gentler detergents, such as octyl glucoside, are used in preference to sodium dodecyl sulfate (SDS). Explain why. 

---