Attached<\/a> 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<\/a><\/p>\n\n\n\n AOT Mixtures<\/strong><\/em><\/p>\n\n\n\n AOT<\/strong><\/p>\n\n\n\n PLEASE NOTE: most proteins encapsulate clearly in AOT reverse micelles but are completely unfolded. CHECK BY NMR Chemical Name: Sodium Bis-(2-ethylhexyl)-sulfosuccinate Making RM sample: once AOT adjusted to the 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; the sample should be mostly clear.<\/p>\n\n\n\n Calculating Water Loading from the 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.<\/p>\n\n\n\n <\/p>\n\n\n\n TAB or DTAB Mixtures<\/strong><\/em><\/p>\n\n\n\n CTAB<\/strong> 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<\/p>\n\n\n\n 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.<\/p>\n\n\n\n Hexanol<\/strong> <\/p>\n\n\n\n DTAB<\/strong> Charge: Positive 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.<\/p>\n\n\n\n 10MAG\/LDAO Mixtures<\/strong><\/em> (these are our preferred surfactants; see Dodveski et al (2014) here<\/a>)<\/p>\n\n\n\n 10MAG<\/strong> 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<\/p>\n\n\n\n Calculating Water Loading from 1H spectrum: Integrate the water peak and the #<\/strong> 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.<\/p>\n\n\n\n LDAO<\/strong>
PLEASE NOTE: The pH needs to be adjusted; Sigma batch generally gives pH ~5. See Marques et al (2014)<\/a> for pH adjustment instructions.<\/strong><\/p>\n\n\n\n
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.<\/p>\n\n\n\n![\"AOT\"\/](\"https:\/\/www.med.upenn.edu\/wandlab\/methods\/images\/AOT.jpg\")
<\/figure>\n\n\n\n
Chemical Name: Cetyltrimethylammonium bromide
Molecular Weight: 364.46 g\/mol
State: White powder
Hexanol Needs: 450\u03bcM 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<\/p>\n\n\n\n
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<\/p>\n\n\n\n![\"CTAB-Hexanol\"\/](\"https:\/\/www.med.upenn.edu\/wandlab\/methods\/images\/CTAB-Hexanol.jpg\")
<\/figure>\n\n\n\n
Chemical Name: Dodecyltrimethylammonium bromide
Molecular Weight: 308.35 g\/mol
State: White powder
Hexanol Needs: Approximately 50\u03bcM more than you would use with a CTAB sample<\/p>\n\n\n\n\n
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).<\/p>\n\n\n\n\n
Chemical Name: Decanoyl-1-rac-glycerol
Molecular Weight: 246.34 g\/mol
State: White powder
Hexanol Needs: Often times none, sometimes need 1-2 \u03bcL (~20\u03bcM) 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<\/a><\/strong>. 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 \u03bcL at a time with vortexing for 30 seconds in between until the sample gets clearer (do not exceed ~ 2\u03bcL)<\/p>\n\n\n\n
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<\/a><\/strong> for adjustment instructions.
Making RM sample: SEE ABOVE
Calculating Water Loading from 1H spectrum: SEE ABOVE.<\/p>\n\n\n\n