Biology Reference
In-Depth Information
1 M TRIS-HCl BUFFER PH 7.0:
7.28 g Tris-HCl salt.
0.47 g Tris base.
Dissolve with purified water and adjust volume to 50 mL.
Filter with 0.2
m Steriflip and store at
20
C.
m
20% CHAPS:
5 g CHAPS.
20 mL purified water.
Dissolve by mixing on a motorized wheel (
30 min).
20
C.
Filter with 0.2
m Steriflip and store at
m
10% DM:
5 g DM.
45 mL purified water.
Dissolve by mixing on a motorized wheel (
30 min).
20
C.
Filter with 0.2
m Steriflip and store at
m
15.6.3
Procedure
Disperse 590 mg CHS-Tris salt (
472 mg CHS) in 10 mL 20% CHAPS (final
concentrations 4.65% CHS and 19.7% CHAPS) on a magnetic stirrer. The
opalescent solution is subjected tomultiple freeze-thaw cycles until optically clear.
The clear solution is filtered with a 0.2
¼
20
C.
Determine the weight of the clean and dry 50 mL glass round flasks using an
analytical balance.
Transfer the 4 mL 25 mg/mL DOPC solution in chloroform to a 50 mL round
flask using a clean glass syringe. Evaporate the solvent under a steam of argon or
nitrogen under a chemical fume hood. Rotate the flask to generate an evenly
thin lipid film. Remove the last traces of chloroform by putting the container on a
vacuum system overnight. Determine the amount of lipid by weighing the
flask and subtraction of the empty weight.
Add 10 mL 10% DM to the flask with 105.5 mg dried DOPC (1.06% final
concentration) and disperse the lipid using a water bath sonicator. The resulting
opalescent solution is cleared by multiple freeze-thaw cycles. The clear
solution is filtered with a 0.2
m
m Steriflip unit and store at
m syringe filter using a glass syringe (or methanol
washed disposable plastic syringe) and stored at
m
20
C.
Determine the weight of the clean and dry 15 mL glass round flasks using an
analytical balance.
Transfer the content of two glass vials with each 25 mg DOPS solution in
chloroform to a 15 mL round flask using a glass Pasteur pipette. Evaporate the
solvent under a steam of argon or nitrogen under a chemical fume hood. Rotate
the flask to generate an evenly thin lipid film. Remove the last traces of
