Biology Reference
In-Depth Information
Methyl viologen dichloride hydrate (Aldrich 856177, MW(anhydrous)
¼
257.16 g/mol, unspecified water content)
D
-(
180.2 g/mol)
Glucose oxidase from
Aspergillus niger
type VII
(Sigma G2133, 195.7 U/mg)
or
CAT: catalase, from
Aspergillus niger
(Sigma C3515, ammonium sulfate
suspension 28.73 mg/mL, 9473 U/mg, 272 U/
þ
)-glucose (Sigma G5767, MW
¼
L)
Ultrafree-MC, centrifugal filtration unit with 0.22
m
m microporous PVDF
m
membrane (Millipore)
Millex-GP syringe filter, 0.22
m, polyethersulfone, 33 mm (Millipore)
5 mL plastic syringe (BD 309646, Fisher)
m
15.8.8.2
Solutions and buffers
200 mM TX:
50 mg Trolox.
950
L DMSO.
Aliquot and store at -80
C for up to 6 months or short-term at -20
C.
(Sensitive to oxidation!)
m
200 mM MV:
25.7 mg methyl viologen dichloride hydrate.
475
L purified water.
Filter with Ultrafree-MC spin filter.
Store at -20
C.
m
GOD:
27.8 mg 195.7 U/mg glucose oxidase (final concentration 10.9 U/
L).
m
150
L purified water.
Mix with pipette to dissolve protein.
Add 322
m
L 78% glycerol (final concentration 50%).
Filter with Ultrafree-MC spin filter.
Store at -20
C.
m
10% GLUCOSE:
500 mg
D
-(
)-glucose.
4.5 mL purified water.
Filter with syringe filter.
Aliquot (500
þ
L) and store at -20
C.
m
15.8.8.3
Procedure
Complex lipid/detergent mixtures may be added to enhance the stability of some
GPCRs (
Knepp et al.
,
2011
); use PBS with 0.1% DM otherwise.
