Biology Reference
In-Depth Information
15.7.2
Step 2: functionalize glass surface with epoxy silane
15.7.2.1
Materials
GOPS, 3-glycidyloxypropyl trimethoxysilane (Aldrich 440167, Dow Z-6040)
Acetic acid, glacial (ACS grade, Fisher)
Ethanol, absolute (200 proof, USP grade)
Purified and filtered water (Milli-Q Synthesis, Millipore)
Steriflip-GP, 0.22
m polyethersulfone (PES) membrane (Millipore)
m
15.7.2.2
Solutions and buffers
GOPS SOLUTION:
500
L acetic acid (1% final concentration, resulting pH
4.5).
m
2.5 mL purified water (5% final concentration).
46 mL absolute ethanol (92% final concentration).
1 mL GOPS (aspirate from Sure/Seal bottle using 22-gauge needle and methanol-
cleaned and air-dried plastic syringe).
Mix on wheel for 15 min.
Filter and degas with Steriflip and use immediately.
15.7.2.3
Procedure
Load 20
L GOPS solution per well of the freshly cleaned 384-well glass-bottom
microplate.
Incubate 60 min at RT.
Over a sink, shake off the GOPS solution from the wells.
Spray the plate evenly with 70% ethanol to wash the wells. Shake off the liquid
into the sink and repeat this ethanol rinse three more times.
Place a regular 384-well plate upside-down on the bench.
Add two sheets of Whatman filter paper cut in 11
m
7.5 cm pieces.
Place the ethanol washed plate upside-down onto the filter papers.
Load this sandwich in a microplate swinging bucket and balance the rotor.
Centrifuge 5 min at 500
g
.
Remove plate from stack and cover it with its lid.
Transfer plate in the vacuum chamber of a SpeedVac and keep it overnight under
high vacuum of a dual-stage oil pump (less than 0.05 Torr/6.7 Pa pressure).
The silanized plate should be used within a day but may be stored in a sealed
plastic bag for a few weeks.
15.7.3
Step 3: immobilization of biotinylated-bovine serum albumin
15.7.3.1
Materials
Biotin-LC-BSA (Pierce 29130, nominal 25 mg, 8 biotin/BSA)
Purified and filtered water (Milli-Q Synthesis, Millipore)
Sodium tetraborate decahydrate (Sigma B9876, MW
¼
381.4 g/mol)
Tris base (Trizma base, Sigma 93362 , MW
¼
121.14 g/mol)
BSA, fatty acid-free (Sigma A8606)
