Biology Reference
In-Depth Information
15.7.3.2
Solutions and buffers
0.2 M BORATE:
0.954 g sodium tetraborate decahydrate (M
381.4, Sigma B9876).
Dissolve with purified water and adjust to 50 mL.
The solution has pH 9.2.
Filter with Steriflip.
¼
1 M TRIS BASE:
6.057 g Tris base.
Dissolve with purified water and adjust to 50 mL.
Filter with Steriflip.
20 mg/mL BIOTINYL-BSA:
30 mg biotin-LC-BSA.
1.5 mL purified water.
Aliquot (250
80
C.
m
L) and store at
5% BSA:
2.5 g BSA, fatty acid-free.
47.5 mL purified water.
Filter with Steriflip.
Aliquot (1 mL per vial) and store at
80
C.
COATING SOLUTION:
250
L 20 mg/mL biotinyl-BSA (final concentration 1 mg/mL).
m
10
L 5% BSA (final concentration 0.1 mg/mL).
2.24 mL H
2
O.
2.5 mL 0.2 M borate.
1.85
m
L 10% DM.
Filter with Steriflip.
m
BLOCKING SOLUTION:
300
L 1 M Tris base.
2.7 mL purified water.
3 mL 0.2 M borate.
Filter with Steriflip.
m
15.7.3.3
Procedure
Load 10
L coating solution per well to columns 2-23 of the silanized
microplate. (On epMotion, use multidispense, change tips before aspiration and
water liquid-type settings.)
Centrifuge 5 min at 500
m
g
.
Seal plate in a ziplock plastic bag with a wet filter paper.
Incubate first 90 min at RT and then overnight at 4
C.
