Biology Reference
In-Depth Information
10% NaN
3
:
100 mg sodium azide
0.9 mL purified water
15.5.2.3
Procedure
Biotinylation of antibody with amino-reactive sulfo-NHS-SS-biotin. Supplement
2 mL 10 mg/mL 1D4 mAb with 200
L 10 mM sulfo-
NHS-SS-biotin (12-fold molar excess over protein); mix for 2 h at RT.
Equilibrate a disposable plastic column with 10 mL packed Sephadex G50 with
30 mL PBS. Load reaction mixture. Elute with PBS and collect 1 mL fractions.
Determine the protein concentration in the fractions by UV-Vis spectroscopy.
L10
PBS. Add 150
m
m
Combine the peak fractions. Supplement 1 mL sample with 148
L 78% glycerol
m
(final concentration 10%) and 2.3
L 10% NaN
3
(final concentration 0.02%) and fil-
m
ter with 0.22
m syringe filter. Snap freeze 200
L aliquots in liquid nitrogen and
m
m
80
C.
store at
15.6
PROTOCOL 4: PREPARATION OF
DETERGENT-SOLUBILIZED LIPIDS
15.6.1
Materials
CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate,
anagrade, MW
¼
614.9 g/mol; Affymetrix)
¼
DM (n-dodecyl-
510.6 g/mol; Affymetrix)
CHS (cholesteryl hemisuccinate (Tris salt), MW
¼
607.9 g/mol; Affymetrix)
DOPC (1,2-dioleoyl-
sn
-glycero-3-phosphocholine, MW
¼
786.1 g/mol; Avanti
Polar Lipids)
DOPS (1,2-dioleoyl-
sn
-glycero-3-phospho-
L
-serine (sodium salt),
MW
β
-D-maltopyranoside Anagrade, MW
¼
810.0 g/mol; Avanti Polar Lipids)
15.6.2
Solutions and buffers
78% GLYCEROL:
10 mL purified water.
35 mL glycerol.
Adjust volume to 45 mL with water.
Filter with 0.2
20
C.
m Steriflip and store at
m
2 M (NH
4
)
2
SO
4
:
13.2 g ammonium sulfate.
35 mL purified water.
Adjust volume to 50 mL with water.
Filter with 0.2
20
C.
m Steriflip and store at
m
