Increasing the critical micelle concentration of triton X-100...

3 posts / 0 new
Last post
JohnRowland
JohnRowland's picture
Increasing the critical micelle concentration of triton X-100...

A plea for intellectual assistance...

I am currently purifying a transmembraneous protein from the outer mitochondrial membrane with the aid of the non-ionic detergent Triton X-100. The working concentration of Triton X-100 I am using is 3% (w/v), which is well over the critical micelle concentration (CMC; ~0.033 %) for this detergent. The existence of such micelles aid in the solubilisation of hydrophobic proteins from membrane interiors, but present problems for further downstream chromatographic and enzymatic characterisation.

Does anyone know of a method I could use to disintegrate the micelles formed, without unnecessary detriment to the solubilised proteins?

I have considered diluting the detergent-solubilised protein solution below the CMC, or adding an organic solvent to perhaps demote the thermodynamic favourability for micelle formation and increase the CMC.

If anyone else has any suggestions that might help I would be extremely grateful. Thanks.

Richard Taylor
Richard Taylor's picture
Some ideas:

Some ideas:

*Drip the micelle suspension into distilled water, this should release the micelle content.

*With sonication you could obtain release of the content of the micelles with the micelles remaining intact making the purification easy. This might be the best option, perhaps dilute a little then sonicate to reduce the chance of reincorporation of the released material into the reforming micelles.

*Slow freeze thaw cycles that make big ice crystals might do it - depends if your protein is sensitive to the process.

*Try a "High hydrostatic pressure" step.

*Membrane disrupting peptides / lipids are an option but add to the purification challenge.

If you're working in a "Biochemistry" environment you might want to seek assistance from someone in Biochemical Engineering.

jachmoody
jachmoody's picture
A short pass through a g15

A short pass through a g15 chromatography column may be a good way to eliminate or reduce the detergent concentration or or a treatment with ultrafiltrationor dialysis.

jim achmoody

Richard Taylor wrote:

Some ideas:

*Drip the micelle suspension into distilled water, this should release the micelle content.

*With sonication you could obtain release of the content of the micelles with the micelles remaining intact making the purification easy. This might be the best option, perhaps dilute a little then sonicate to reduce the chance of reincorporation of the released material into the reforming micelles.

*Slow freeze thaw cycles that make big ice crystals might do it - depends if your protein is sensitive to the process.

*Try a "High hydrostatic pressure" step.

*Membrane disrupting peptides / lipids are an option but add to the purification challenge.

If you're working in a "Biochemistry" environment you might want to seek assistance from someone in Biochemical Engineering.