Nanodisc Formation

More information about nanodiscs: http://sligarlab.life.uiuc.edu/nanodisc.html)
(Modified from a protocol originally developed by Saskia Neher, and provided by David Booth.)

Materials:

*** Dried lipid film (e.g. DMPC) in a glass tube, total 20 uMole (before drying: 100 mM in 200 ul chloroform)
*** Sodium cholate stock in dH2O, 100 mM
*** Buffer A: 20mM Tris, pH7.5, 100 mM NaCl (can be made from a 20x stock)
*** Buffer B: Buffer A plus 80 mM cholate (made freshly)

Procedure:

(1) Solubilize the dried lipid film in 0.5 ml buffer B, by sonication and warming up,
so that [lipid]=40 mM. Make sure [cholate]:[lipid]= 2:1
(2) Mix everything in 100 ul reaction volume. Make the final [cholate]= 12-40 mM
e.g. stock [MSP1D1]= 263.1 uM; using DMPC, go with lipid:protein= 80:1
Final [MSP1D1]= 100 uM: 100 uM x 100 ul / 263.1 uM = 38 ul
Final [DMPC] = 8 mM: 8 mM x 100 ul / 40 mM = 20 ul

Mixture:

Buffer A : 37 ul
Buffer B (80 mM cholate) : 5 ul (gives 4 mM cholate)
MSP1D1 (263.1 uM) : 38 ul
DMPC (40 mM lipid/80 mM cholate) : 20 ul (gives 16 mM cholate)

(3) Incubate at appropriate temperature for 15-60 min on a rotator
DPPC: 37 C, DMPC: 25 C, POPC: 4 C
(4) Take 200 ul Biobeads into a tube, wash with ddH20 for several times by inverting and quick spin
(5) Add the nanodiscs formation mixture into the washed Biobeads, and incubate at room temperature
on a rotator for 4hrs to overnight
(6) The suspension is collected, filtered and fractionated using FPLC.