Reverse Micelle Surfactants

See Nucci et al. (2014) J. Magn. Reson. for a recent review of this topic.

Attached is an excel sheet that helps aid in your surfactant screening. Anything that is in blue is editable. This is useful when optimizing RM conditions since it limits the amount of math that you need to do. However, make sure you know how to do all of the math in the spreadsheet or visit RM Introduction

AOT Mixtures

AOT

PLEASE NOTE: most proteins encapsulate clearly in AOT reverse micelles but are completely unfolded. CHECK BY NMR
PLEASE NOTE: The pH needs to be adjusted; Sigma batch generally gives pH ~5. See Marques et al (2014) for pH adjustment instructions.


Chemical Name: Sodium Bis-(2-ethylhexyl)-sulfosuccinate
Molecular Weight: 444.56 g/mol
State: Sticky Solid below pH 8, grainy powder above
Hexanol Requirement: NONE
Charge: Negative
pH Considerations: AOT is pH sensitive with a pKa around 5.

Making RM sample: once AOT adjusted to desired pH, AOT is readily soluble in pentane. Dissolve the proper amount of AOT in your desired amount of low viscosity solvent and add the aqueous phase. Vortex for ~30 seconds; sample should be mostly clear.

Calculating Water Loading from 1H spectrum. Integrate the water peak and the starred multiplet as shown below. Set the integral for the multiplet to two. Divide the new water integral by two for a final water loading.

AOT


CTAB or DTAB Mixtures

CTAB
Chemical Name: Cetyltrimethylammonium bromide
Molecular Weight: 364.46 g/mol
State: White powder
Hexanol Needs: 450μM works well with 75mM surfactant (double the hexanol for double the surfactant). Slight changes in hexanol can significantly alter the stability of a sample. More hexanol is needed for high pressure samples.
Charge: Positive
pH Considerations: NONE

Making RM sample: CTAB is not readily soluble in pentane. Dissolve the proper amount of CTAB in your desired amount of low viscosity solvent and add the proper amount of hexanol. Vortex for ~15 seconds; then add the aqueous phase and vortex for upwards of 30 seconds (vortex until as clear as possible). Oftentimes the mixture will remain cloudy no matter how much vortexing you do, this is fine. Spin down the RM mixture at room temperature for ~ 3 minutes and transfer your sample without disturbing the pellet

Calculating Water Loading from 1H Spectrum: Integrate the water peak and the starred singlet as shown below. Set the integral for the singlet to nine. Divide the new water integral by two for a final water loading.

Hexanol
Chemical Name: n-hexanol
Molecular Weight: 102.18 g/mol (0.82 g/ml density liquid)
State: Clear, slightly viscous liquid (~8M concentration)
Charge: None
pH Considerations: NONE

CTAB-Hexanol


DTAB
Chemical Name: Dodecyltrimethylammonium bromide
Molecular Weight: 308.35 g/mol
State: White powder
Hexanol Needs: Approximately 50μM more than you would use with a CTAB sample

Charge: Positive
pH Considerations: NONE Making RM sample: DTAB is not readily soluble in pentane. Dissolve the proper amount of DTAB in your desired amount of low viscosity solvent and add the proper amount of hexanol. Vortex for ~15 seconds; then add the aqueous phase and vortex for upwards of 30 seconds (vortex until as clear as possible).

Calculating Water Loading from ZGPR: Integrate the water peak and the starred singlet as shown below. Set the integral for the singlet to nine. Divide the new water integral by two for a final water loading.

DTABpicture.jpg


10MAG/LDAO Mixtures (these are our preferred surfactants; see Dodveski et al (2014) here)

10MAG
Chemical Name: Decanoyl-1-rac-glycerol
Molecular Weight: 246.34 g/mol
State: White powder
Hexanol Needs: Often times none, sometimes need 1-2 μL (~20μM) to make sample stable. More hexanol is needed for high pressure samples i.e. those prepared in butane/propane/ethane.
Charge: Neutral
pH Considerations: NONE
Making RM sample: 10MAG is often used in conjunction with LDAO (see below) in a ratio that has more 10MAG than LDAO (usually somewhere between 60:40 and 70:30). DTAB or CTAB in small amounts (~5%) is sometimes used in conjunction with LDAO/10MAG. Sometimes LDAO is completely replaced by one of the positive surfactants (CTAB or DTAB). If RM made with LDAO, pH considerations need to be taken into account: see pH in Reverse Micelles. Once you have pH adjusted powder, dissolve in the proper amount of low viscosity solvent, then add the aqueous phase. If the sample is clear after vortexing at this point, stop. If not, then add hexanol 0.5 μL at a time with vortexing for 30 seconds in between until the sample gets clearer (do not exceed ~ 2μL)

Often the mixture will remain cloudy no matter how much vortexing you do or hexanol you add, this is fine. Spin down the RM mixture at room temperature for ~ 3 minutes and transfer your sample without disturbing the pellet

Calculating Water Loading from 1H spectrum: Integrate the water peak and the # LDAO singlet as shown below. Set the integral for the singlet to six. Multiply the new water integral by the molar fraction of LDAO then divide by two for a final water loading.

LDAO
Chemical Name: Lauryldimethylamine-N-oxide
Molecular Weight: 229.41 g/mol
State: Sticky White powder
Hexanol Needs: SEE ABOVE
Charge: Zwitterionic
pH Considerations: LDAO is pH sensitive with a pKa around 4. pH needs to be adjusted; Affymetrix batch is around pH 7. See pH in Reverse Micelles for adjustment instructions.
Making RM sample: SEE ABOVE
Calculating Water Loading from 1H spectrum: SEE ABOVE.

LDAO10MAGpicture.jpg


"Triple Surfactant" Mixtures (C12E4)

The polyether series such as C12E4 are used to dilute charge from the surfactant shell that may be causing the encapsulated protein to interact with the reverse micelle surface. This sometimes occurs with highly charged proteins such as cytochrome c. See here for a detailed dscription. Because of the double layer of charge (i.e. including surfactant counterions) this is often difficult to predict but charge dilution and balancing helps alleviate it when it does. The downside is that these species are long and increase the diameter of the reverse micelle particle thereby slowing tumbling.

C12E4picture.jpg