Biology Reference
In-Depth Information
15.7.4.3 Procedure
￿ Select block of wells used for one experiment, for example, 4 columns and
12 rows (wells C3-N14). Mark the lid to keep track of used wells.
￿ Load 10
L priming solution to each well.
￿ Aspirate wells.
￿ Load 10
m
L NeutrAvidin solution to each well. (On epMotion with TS-50 single
channel tool, use multidispense, change tip before each aspiration and water
liquid-type settings.)
￿ Inspect wells for incomplete wetting and centrifuge if necessary.
￿ Incubate 15 min at RT.
￿ Aspirate wells.
￿ Load 10 m L blocking solution to each well.
￿ Incubate 10 min at RT.
￿ Proceed with next step, or store at 4 C for up to a week.
m
15.7.5 Step 5: immobilization of biotinylated antibody
15.7.5.1 Materials
D -Biotin (Invitrogen B-1595, MW
¼
244.31 g/mol)
1.0 M KOH (volumetric, Fluka)
15.7.5.2 Solutions and buffers
0.2 M BIOTIN:
48.9 mg D -biotin.
951
L 1.0 M KOH.
Aliquot (50
m
80 C.
L) and store at
m
1 mM BIOTIN:
5
L 0.2 M biotin
995
m
L PBS
m
BIOTINYLATED ANTIBODY SOLUTION:
10
m
g biotinylated 1D4 (e.g., 1.84
m
L 5.42 mg/mL 1D4-ss-biotin)
40
L 5% BSA
10 m L 0.37% DM
950
m
L PBS
m
BIOTIN SOLUTION:
3
L 1 mM biotin (3
M final concentration)
m
m
40
m
L 5% BSA
10
m
L 0.37% DM
947
m
L PBS
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