Alignment Sample Preparation
Phage
Start with a protein stock 1-2 mM and a pf1 phage stock of 50 mg/mL. Prepare a sample of 10 mg/mL. Prepare a sample of 10 mg/mL of phage as follows:
250 uL protein 175 uL protein
90 uL phage 50 uL phage
45 uL D2O 25 uL D2O
65 uL buffer
total volume 450 uL total volume 250 uL
Collect 15N-1H RDCs and determine level of alignment. Target range is at least 20 Hz, which will roughly correspond to a 2H splitting of 13 Hz. If sample is not aligned strongly enough, add phage to a concentration of 15 mg/mL and repeat.
Peg
Start with a protein stock of 1-2 mM that contains 10% D2O and a peg bicelle stock of ~16%
50 uL C12E5 (pentaethylene glycol monododecyl ether)
16 uL Hexanol
250 uL Buffer containing 10% D2O at correct pH (200 uL buffer +50 uL D2O)
Mix C12E5 and buffer well with vortexing. Add hexonal in 4 uL increments, vortexing well after each addition, Solution goes from clear to milky, turbid, then to translucent and viscous with lots of bubbles. Continue adding hexanol until solution goes clear again. If it becomes milky/turbid again, you have gone past the nematic phase.
Prepare a sample of 4-6% peg by diluting protein and peg (65% protein and 35% peg) with vortexing. Let sample sit at room temperature overnight to allow equilibration of alignment. 2H splitting peaks should be symmetrical and of equal height. There should be no residual isotropic peak. Collect 15N-1H RDCs and determine level of alignment. Target range is at least 20 Hz, which will roughly correspond to a 2H splitting of 10-16 Hz.
Peg/CTAB
Start with a protein stock of 1-2 mM that contains 10% D2O and a peg bicelle stock of 16%.
50 uL C12E5 (pentaethylene glycol monododecyl ether)
16 uL Hexanol
250 uL Buffer containing 10% D2O at correct pH
Mix C12E5 and buffer well with vortexing. Add hexonal in 4 uL increments, vortexing well after each addition, Solution goes from clear to milky, turbid, then to translucent and viscous with lots of bubbles. Continue adding hexanol until solution goes clear again. If it becomes milky/turbid again, you have gone past the nematic phase.
Prepare a sample of 4-6% peg by diluting protein and peg (65% protein and 35% peg) with vortexing. Let sample sit at room temperature overnight to allow equilibration of alignment. 2H splitting peaks should be symmetrical and of equal height. There should be no residual isotropic peak. Collect 15N-1H RDCs and determine level of alignment. Target range is at least 20 Hz, which will roughly correspond to a 2H splitting of 10-16 Hz.
Peg can be doped with CTAB to provide a second alignment tensor. The ratio of Peg:CTAB is 27:1.
Peg can also be doped with SOS to provide another alignment tensor. The Peg-SOS is now negative charged.
Bicelle
Start with a protein stock of 1-2 mM that contains 10% D2O and a bicelle stock of 15% (ether bicelles hydrolysis resistant).
62.2 mg DMPC
12.7 mg DHPC
Mix each lipid separately in 300 uL buffer containing D2O. Vortex thoroughly. DHPC should dissolve easily. DMPC will not dissolve but rather forms a milky white suspension. When both samples are homogeneous, add DHPC to DMPC and vortex well. Smal white chunks may form. Freeze with liquid N2 or dry ice/acetone bath. Thaw to RT, vortex. Sonicate. Repeat at least three times and continue process until sample is clear at RT with no chunks. If chunks remain, place bicelles in an -80 C freezer 1-2 hours, thaw, vortex.
Dissolve to 3.5-4.5% with protein solution. Sample should be isotropic at 25 C and aligned from 25 -30 C. 2H splitting peaks should be symmetrical and of equal height. There should be no residual isotropic peak. Collect 15N-1H RDCs and determine level of alignment. Target range is at least 20 Hz, which will roughly correspond to a 2H splitting of 10-16 Hz.